Linked ligation

ABSTRACT

The invention generally relates to capturing, amplifying, and sequencing nucleic acids. In certain embodiments, copies of the sense and antisense strands of a duplex template nucleic acid are captured using linked capture probes and multiple binding and extension steps to improve specificity over traditional single binding target capture techniques. Methods of seeding sequencing clusters with sense and antisense strands of a target nucleic acid are also disclosed including identifying the strands using sense-specific barcodes and confirming base calls using two sense-specific sequencing reads. Linked adapters may be used to increase adapter ligation selectively or efficiency and yield.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Nos. 62/432,277 filed on Dec. 9, 2016, and 62/569,824 filedOct. 9, 2017, both of which are incorporated by reference herein.

FIELD OF THE INVENTION

The invention generally relates to capturing, amplifying, and sequencingnucleic acids.

BACKGROUND

High-throughput genomic sequencing platforms generate large amounts ofdata at affordable prices, but they are not sufficiently accurate. Eventhe best sequencing techniques have error rates around 1 percent. Thattranslates to hundreds of thousands of errors in the sequence of asingle human genome. Inaccurate base calling leads to sequencemisalignment and the misidentification of mutations. Although basecalling and alignment algorithms are available, quality is negativelyimpacted by amplification and sequencing errors.

Current methods of isolating target nucleic acids from a sample forsequencing are complicated and can benefit from increased accuracy.Additionally, once target nucleic acids are captured and sequenced, basecalling and alignment remain riddled with errors. For example, in thecurrently leading sequencing platform, DNA fragments are attached to asolid support, such as a channel wall. Once a fragment is attached tothe solid support, the fragment is amplified and the amplificationproducts attach to the solid support proximate to the seeding fragment.The process repeats until a cluster of amplification products thatshould be identical to the seeding fragments forms. However, only onefragment seeds a cluster. If there is an error in the seeding fragment,or an error is made in the amplification of the cluster the error isrepeated in the all or part of cluster. This error leads tomisidentifying a base and complicating sequencing alignment.

To catch these types of errors, standard barcode sequencing methods usetens to hundreds of copies of the same template, or ten to hundreds ofclusters to create a sample pool for comparison. By drasticallyincreasing the number of copies or clusters, an error can be determined.However, this strategy is expensive and consumes sequencing bandwidth.

SUMMARY

The invention provides linked ligation adapters and methods allowing forincreased ligation yields and simplified workflows in many capture andsequencing techniques. By linking sequencing or universal priming siteadapters to sequence specific probes, target sequence selection andcapture can be combined with adapter ligation to reduce steps andincrease target selectivity. Target specific probes bring adapterslinked thereto into close proximity to the target sequence at whichpoint the linked adapters may be ligated to the target sequence. Becauseadapters are selectively ligated to the target sequence, subsequentamplification with universal primers complimentary to sites in theligated adapters will only amplify the target sequence, preparing atargeted library ready for sequencing. Linked ligation techniques may beused to capture nucleic acid fusions where only one side of thebreakpoint is known. By linking the adapters to sequence specific probescomplimentary to the known portion of the fusion, methods may still beused to selectively ligate adapters and amplify only the target fusionnucleic acid for sequencing. In certain embodiments, one of the linkedligation adapter and probe molecules may be bound to a flow cell suchthat target nucleic acids may be captured and prepared for flow cellamplification or sequencing through adapter ligation at the same time,simplifying existing workflows.

Methods of the invention contemplate double stranded linked ligation. Byusing isothermal recombinase and single stranded binding proteins togenerate strand invasion of double stranded DNA (dsDNA) with theligation probe (similar to Recombinase Polymerase Amplification (RPA))methods provide targeted ligation of adapters onto dsDNA.

The invention provides linked ligation adapters and methods allowing forligation of two different adapters to a single DNA template, andincreased ligation yields. By linking two different adapters prior toligation, reaction kinetics are such that once one adapter is ligated,the second different (linked) adapter is brought into close proximity tothe unligated end of the DNA for subsequent ligation. Linked adapterligation may be used on single or double stranded DNA, in applicationswhere it is desirable to ensure two different adapters are ligated toeach template.

The invention provides methods of linked target capture for singlestranded or duplex DNA molecules. Solution-based target capture methodsas well as droplet-based target capture methods are provided. Thesolution and droplet based methods use linked target capture probesincluding a universal probe and a target specific probe wherein thereactions occur under conditions that require the target specific probeto bind in order to permit binding of the universal probe. Becausemultiple binding and extension steps are involved, specificity isimproved over traditional single binding target capture. The bounduniversal probe is then extended using strand displacing polymerase toproduce copies of the target strands which can then be amplified usingPCR with universal primers. Methods of the invention replacePCR-capture-PCR workflows with a single PCR and capture step. Linkedcapture probes can be used in one or both senses of DNA where higherspecificity and duplex information are required. Multiple linker typesare possible as discussed below. Similar to solution-based targetcapture methods of the invention provide for droplet based methods thatallow a user to perform target capture in droplets, rather than beingrestricted to multiplexed PCR in droplets. Capture methods may becombined with linked primers as described herein to create linked,duplex molecules from droplets. In certain embodiments, nanoparticlescomprising target capture probes as well as universal primers can beused to capture targeted regions from a pool of 5′-linked molecules,converting only the targeted molecules into duplex seeds for sequencingclusters.

The invention also provides methods for increasing base calling accuracyby physically linking fragments representing both the sense andantisense strands of a duplex DNA molecule. By linking both strands intoa single read, information density is increased and error rates arereduced as the duplex data permits ready differentiation between truevariants and errors introduced in amplification or sequencing (e.g.,errors that a polymerase might make in one sense are not likely to berepeated in both strands while a true variant would be). Sense specificbarcodes may be used to confirm the presence of both sense and antisensetemplate copies in a cluster. Dedicated sense and antisense sequencingreads may be used to differentiate between introduced errors and truevariants.

Methods of the present invention have applications in sample preparationand sequencing. In sample preparation methods, the present inventionallows for fragments of both sense and antisense strands of a duplexnucleic acid to be joined together. A linking molecule joins thefragments, creating a complex. The complex can include adapters,primers, and binding molecules in addition to at least the two strands.In samples having low target DNA content such as cancer samples, bylinking both strands together, fragments can be amplified and sequencedwith increased accuracy with ready identification of sequencing andamplification errors.

Linked fragments may be created through amplification of a nucleic acidfragment with linked amplification primers. In certain embodiments,universal priming sites may be ligated onto the target fragment tocreate a template molecule. Methods may include droplet and non-dropletworkflows and produce linked molecules representing both strands atabout at least a 50% rate. In droplet amplification methods, thetemplate molecule may be added to a droplet along with multiplexed genespecific forward and reverse amplification primers and linked universalprimers. The droplet can then be subjected to emulsion or digital PCRamplification. The amplified products should be linked copies of thesense and antisense strands of the original fragment. Two or moreprimers or nucleic acid fragments may be linked by a polyethylene glycolderivative, an oligosaccharide, a lipid, a hydrocarbon, a polymer, or aprotein. In certain embodiments, four or more biotinylated primers ornucleic acid fragments may be linked with a streptavidin molecule, or afunctionalized nanoparticle. Linked primers of the invention may alsoinclude unique cluster identifier sequences to ensure that all clusterreads originate from the same template molecule.

Methods of the invention include duplex identification strategies fordroplet formed linked duplex molecules. As noted, droplet based methodsof the invention may result in at least a 50% rate of linked duplexfragment formation (linked molecules that contain representations fromeach side of the DNA duplex) so, identification of those productsbecomes important in order to omit data from non-duplex products andreap the accuracy increasing benefits of the duplex products. Duplexidentification methods may include, for example, a two-stage PCRapproach using two sets of primers with different annealing temperatureswhere several initial cycles are performed at low temperature withgene-specific barcoding primers to amplify and identify each sense ofthe duplex, while adding a universal tail for subsequent cycles. Thenumber of barcoding cycles is limited to prevent labeling each sense ofthe duplex with multiple barcodes.

Subsequent cycles may then be performed at high temperature viauniversal primers because the barcoding primers are unable to bind underthose conditions. Duplex products may then be identified by the presenceof their sense specific barcodes during sequencing analysis anddistinguished from non-duplex clusters. The higher fidelity of duplexcluster seeding can therefore be appreciated.

In non-droplet embodiments, a single amplification cycle may be used tocreate a linked duplex molecule having both the sense and antisensestrands of the original fragment. The linked duplex molecule may then bedirectly loaded in a flow cell for sequencing, thereby avoidingamplification induced sequence or length biases or (e.g., in wholegenome sequencing) as well as avoiding amplification introduced errorsand nucleic acid losses from poor loading efficiency. For example, whereloading efficiency of a sequencer can be defined as: (number of outputreads)/ (number of input molecules able to form reads), the loadingefficiency for the Illumina MiSeq is <0.1%, and is similar for otherIllumina instruments. This is largely due to fluidic losses, since over600 uL of sample is loaded into the sequencer, while only ˜7 uL isretained inside the flow cell for binding, resulting in large losses ofstarting material. The non-droplet, direct load methods described hereinremedy these inefficiencies.

Linked duplex molecule formation may be created by ligating linkingadapters to template molecule and extending with strand displacingpolymerase to create a linked duplex template with sequencing adapters.In various embodiments, the linking adapters may be linked to an adapterto be ligated to the other end of the template to help ensure that twodifferent adapters are ligated to each molecule, nearly eliminatingmolecules having two of the same adapters ligated thereto. The linkedadapters may include a single linking adapter or may be made up of twolinked linking adapters. Linked ligation techniques may be applied toY-adapters and hairpin adapters as well. Ligation efficiency is improvedbecause binding of one adapter increases the likelihood that the second,linked adapter will bind to the other end of the template molecule.Linkers may comprise PEG, nucleotides, inverted nucleotides, or any of avariety of molecular spacers and linkers known in the art. Linkers maybe cleavable (e.g., through UV exposure, uracil, or other digestion) orbe bound together through complementary sequences having a bindingaffinity allowing for denaturing at a selected temperature (T_(m))allowing for release of the link after ligation.

For direct loading embodiments as well as other applications where theyield of flow cell loading and target capture yield are important, itmay be beneficial to combine flow cell loading with targeted sequencing,to minimize loss. Such a combination additionally simplifies theworkflow by eliminating an extra step. While methods exist for targetcapture on the flow cell, they suffer from at least two downsides.First, they are not able to sequence the region that is captured on theflow cell. For short fragments such as cell free DNA, this can amount toa large loss of signal. Secondly, they are unable to capture linkedduplex molecules, as described in the invention, for sequencing.Accordingly, methods of the invention include flow cell based targetcapture of duplex molecules. According to methods of the invention, theflow cell contains one sense of oligos having target regions, while theother sense are hair-pinned and not immediately available for binding.After one sense of linked molecules is captured on the flow cell, theother flow cell oligos are activated to capture the other sense of thelinked fragments (e.g., using a uracil digest, enzyme digestion, orlight). The template may then be extended and cluster generation maycontinue as normal.

Methods of the present invention improve base calling when incorporatedinto amplification techniques. In traditional amplification methods,amplicons are created from a single template. If an error exists in thefragment, the error is propagated through the amplification products.Instead of using a single template, multiple templates (representingeach sense of a duplex nucleic acid fragment) are used to create theamplification products. In the event that there is an error developed inone of the strands, the use of both strands, as opposed to a single one,allows such an error to be identified at the sequencing step anddifferentiated from true variants which are likely to be found in bothstrands. In certain techniques of the invention, by seeding withmultiple templates, errors can be differentiated from true variantsthrough a drop in sequencing quality in a single read at the positionwhere the bases are not the same (a true variant would be present on allreads, providing a strong signal). In embodiments seeding a cluster witha sense and antisense strand, true variants and errors may be identifiedby comparing results of a first sense read to a second antisense read toconfirm the presence of the variant on both template strands.

Methods of the present invention improve amplification on a solidsupport, such as in the Illumina platform (Illumina, Inc. San Diego,Calif.) or the Ion Torrent platform (Thermo Fisher Scientific Inc.,Waltham,. Mass.). In the Illumina technique, using bridge amplification,clusters of amplicons are formed. If an error exists in the fragment,the error is repeated in the cluster. However, with the presentinvention, linked duplex fragments are contacted to the solid support.The fragments representing both sense and antisense strands of theoriginal duplex molecule seed the cluster, resulting in a fraction ofthe total amplicons being derived from each of the strand fragments.This technique allows for an error to be readily determined at thesequencing step as well as aiding in calling true variants anddifferentiating them from sequencing or amplification (e.g., PCR)errors.

Methods of the invention improve multiplexing amplification processes.In some embodiments of the present invention, linked fragments can beformed in or introduced into a droplet for subsequent amplification. Ifan error exists in some of the fragments, the error is determinable withthe raw sequencing data. In some embodiments, the linked fragments canbe bound to a microsphere and then with amplification, the fragmentsseed the microsphere with amplicons. By providing the advantage offorming a plurality of amplicons using multiple copies of the samefragment, the present invention improves base calling in a variety ofapplications.

Methods of the invention can be incorporated into multiple sequencingplatforms. For example, in traditional sequencing by synthesis, eachbase is determined sequentially. An error is not determined untilbioinformatics techniques are used to analyze the data. However, thepresent invention allows for duplex fragments of nucleic acids to belinked together during sequencing methodologies. By analyzing multiplefragments simultaneously, agreement between the bases indicatesaccuracy, while disagreement between the bases would signal an error.With the present invention, errors are determinable from the rawsequencing data, without the application of bioinformatics. Thistechnique uses fewer copies or clusters, increases sequencingthroughput, and decreases costs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a droplet based method of the invention for creatinglinked duplex nucleic acids.

FIG. 2 depicts an exemplary linked primer and forward and reverse genespecific primers and their use according to one method of the invention.

FIG. 3 depicts an exemplary linked primer and forward and reverse genespecific primers and their use according to one method of the invention.

FIG. 4 shows gene specific primers of the invention.

FIG. 5 depicts a sequencing method of the invention with productsmethods shown in FIG. 2.

FIG. 6 depicts a sequencing method of the invention with productsmethods shown in FIG. 3.

FIGS. 7A and 7B show a non-droplet linked duplex formation method usingone linking adapter.

FIGS. 8A and 8B show a non-droplet linked duplex formation method usingtwo linking adapters.

FIGS. 9A and 9B illustrate steps of a direct loading sequencing methodusing linked duplex molecules.

FIG. 10 shows exemplary steps of a flow cell binding method.

FIG. 11 depicts an exemplary off-line flow cell preparation protocol.

FIG. 12 illustrates flow cell based target capture methods for duplexmolecules

FIGS. 13A-13E depict steps in an exemplary flow cell based targetcapture and sequencing method for duplex molecules.

FIGS. 14A-14D illustrate duplex identification methods according tocertain embodiments.

FIG. 15 shows examples of possible configurations of adapters andprimers.

FIG. 16 shows sequencing error rates using singly seeded clusters thataligned to a KRAS amplicon.

FIG. 17 depicts a singly seeded cluster of the sequencing methods usedto produce the results in FIG. 16 and a doubly seeded cluster of thesequencing methods used in FIG. 29.

FIG. 18 illustrates a base calling method of the invention based on asingle sequencing read and signal quality.

FIG. 19 shows a base calling method based on a comparison of a sense andanti-sense sequencing read.

FIG. 20 illustrates exemplary methods of linked target capture of duplexnucleic acids.

FIG. 21 illustrates amplification methods of linked target capturednucleic acids.

FIG. 22 shows methods of droplet based target capture and linked duplexnucleic acid production.

FIGS. 23A and B show steps of a droplet-based target capture method ofthe invention.

FIG. 24 shows a nanoparticle having universal primers and a strandcomprising a target region complementary to a capture region of thelinked molecule to be captured.

FIG. 25 illustrates binding of the capture region to the target region.

FIG. 26 shows binding of the universal primers to universal primer siteson the linked molecule.

FIG. 27 shows universal primer extension by strand displacing polymeraseto produce nanoparticle linked copies of the target molecule comprisingboth strands of the original linked molecule.

FIG. 28 shows a doubly seeded nanoparticle that may be used to seed acluster on a flow cell sequencer as described elsewhere in theapplication.

FIG. 29 shows sequencing error rates using doubly seeded clusters thataligned to a KRAS amplicon.

FIG. 30 shows exemplary steps of solution-based linked ligation.

FIG. 31 shows linked ligation of adapters to fusion nucleic acids.

FIGS. 32A and 32B illustrate steps of a linked ligation surface capturetechnique according to certain embodiments.

FIGS. 33A-33C illustrate linked adapter ligation according to certainembodiments.

FIG. 34 shows an exemplary configuration of linked adapters includingone linking adapter.

FIG. 35 shows one exemplary configuration of linked adapters includingtwo linking adapters.

FIG. 36 shows another exemplary configuration of linked adaptersincluding two linking adapters.

FIG. 37 shows an exemplary configuration of linked Y-adapters.

FIGS. 38A-D show exemplary methods of double stranded linked ligation.FIG. 38A shows creation of a ligation complex generation of strandinvasion of a genomic template using the ligation complex and singstranded binding protein. FIG. 38B shows subsequent targeted doublestranded ligation of the ligation adapter to the genomic template. FIG.38C shows the results of various second ligations performed on theopposite end of the genomic template. FIG. 38D shows PCR amplificationperformed on the ligated genomic template using adapter-specificprimers.

DETAILED DESCRIPTION

Methods and compositions of the invention include linked adapterligation for improving ligation efficiency and target sequence capturewhile simplifying sequencing workflows. In certain embodiments, theinvention relates to methods for amplifying and sequencing nucleic acidsby joining both strands of a duplex nucleic acid fragment. The use ofboth strands reduces error rates, increases efficiency in alignment, andreduces sequencing costs.

Nucleic acid generally is acquired from a sample or a subject. Targetmolecules for labeling and/or detection according to the methods of theinvention include, but are not limited to, genetic and proteomicmaterial, such as DNA, genomic DNA, RNA, expressed RNA and/orchromosome(s). Methods of the invention are applicable to DNA from wholecells or to portions of genetic or proteomic material obtained from oneor more cells. Methods of the invention allow for DNA or RNA to beobtained from non-cellular sources, such as viruses. For a subject, thesample may be obtained in any clinically acceptable manner, and thenucleic acid templates are extracted from the sample by methods known inthe art. Generally, nucleic acid can be extracted from a biologicalsample by a variety of techniques such as those described by Maniatis,et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y., pp. 280-281, 1982), the contents of which are incorporated byreference herein in their entirety.

Nucleic acid templates include deoxyribonucleic acid (DNA) and/orribonucleic acid (RNA). Nucleic acid templates can be synthetic orderived from naturally occurring sources. Nucleic acids may be obtainedfrom any source or sample, whether biological, environmental, physicalor synthetic. In one embodiment, nucleic acid templates are isolatedfrom a sample containing a variety of other components, such asproteins, lipids and non-template nucleic acids. Nucleic acid templatescan be obtained from any cellular material, obtained from an animal,plant, bacterium, fungus, or any other cellular organism. Samples foruse in the present invention include viruses, viral particles orpreparations. Nucleic acid may also be acquired from a microorganism,such as a bacteria or fungus, from a sample, such as an environmentalsample.

In the present invention, the target material is any nucleic acid,including DNA, RNA, cDNA, PNA, LNA and others that are contained withina sample. Nucleic acid molecules include deoxyribonucleic acid (DNA)and/or ribonucleic acid (RNA). Nucleic acid molecules can be syntheticor derived from naturally occurring sources. In one embodiment, nucleicacid molecules are isolated from a biological sample containing avariety of other components, such as proteins, lipids and non-templatenucleic acids. Nucleic acid template molecules can be obtained from anycellular material, obtained from an animal, plant, bacterium, fungus, orany other cellular organism. In certain embodiments, the nucleic acidmolecules are obtained from a single cell. Biological samples for use inthe present invention include viral particles or preparations. Nucleicacid molecules can be obtained directly from an organism or from abiological sample obtained from an organism, e.g., from blood, urine,cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue.Any tissue or body fluid specimen may be used as a source for nucleicacid for use in the invention. Nucleic acid molecules can also beisolated from cultured cells, such as a primary cell culture or a cellline. The cells or tissues from which template nucleic acids areobtained can be infected with a virus or other intracellular pathogen.In addition, nucleic acids can be obtained from non-cellular ornon-tissue samples, such as viral samples, or environmental samples.

A sample can also be total RNA extracted from a biological specimen, acDNA library, viral, or genomic DNA. In certain embodiments, the nucleicacid molecules are bound as to other target molecules such as proteins,enzymes, substrates, antibodies, binding agents, beads, small molecules,peptides, or any other molecule and serve as a surrogate for quantifyingand/or detecting the target molecule. Generally, nucleic acid can beextracted from a biological sample by a variety of techniques such asthose described by Sambrook and Russell, Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor, N.Y. (2001). Nucleic acidmolecules may be single-stranded, double-stranded, or double-strandedwith single-stranded regions (for example, stem- and loop-structures).Proteins or portions of proteins (amino acid polymers) that can bind tohigh affinity binding moieties, such as antibodies or aptamers, aretarget molecules for oligonucleotide labeling, for example, in droplets.

Nucleic acid templates can be obtained directly from an organism or froma biological sample obtained from an organism, e.g., from blood, urine,cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Ina particular embodiment, nucleic acid is obtained from fresh frozenplasma (FFP). In a particular embodiment, nucleic acid is obtained fromformalin-fixed, paraffin-embedded (FFPE) tissues. Any tissue or bodyfluid specimen may be used as a source for nucleic acid for use in theinvention. Nucleic acid templates can also be isolated from culturedcells, such as a primary cell culture or a cell line. The cells ortissues from which template nucleic acids are obtained can be infectedwith a virus or other intracellular pathogen. A sample can also be totalRNA extracted from a biological specimen, a cDNA library, viral, orgenomic DNA.

A biological sample may be homogenized or fractionated in the presenceof a detergent or surfactant. The concentration of the detergent in thebuffer may be about 0.05% to about 10.0%. The concentration of thedetergent can be up to an amount where the detergent remains soluble inthe solution. In a preferred embodiment, the concentration of thedetergent is between 0.1% to about 2%. The detergent, particularly amild one that is non-denaturing, can act to solubilize the sample.Detergents may be ionic or nonionic. Examples of nonionic detergentsinclude triton, such as the Triton X series (Triton X-100t-Oct-C6H4—(OCH2—CH2)xOH, x=9-10, Triton X-100R, Triton X-114 x=7-8),octyl glucoside, polyoxyethylene(9)dodecyl ether, digitonin, IGEPALCA630 octylphenyl polyethylene glycol, n-octyl-beta-D-glucopyranoside(betaOG), n-dodecyl-beta, Tween 20 polyethylene glycol sorbitanmonolaurate, Tween 80 polyethylene glycol sorbitan monooleate,polidocanol, n-dodecyl beta-D-maltoside (DDM), NP-40 nonylphenylpolyethylene glycol, C12E8 (octaethylene glycol n-dodecyl monoether),hexaethyleneglycol mono-n-tetradecyl ether (C14EO6),octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG), Emulgen, andpolyoxyethylene 10 lauryl ether (C12E10). Examples of ionic detergents(anionic or cationic) include deoxycholate, sodium dodecyl sulfate(SDS), N-lauroylsarcosine, and cetyltrimethylammoniumbromide (CTAB). Azwitterionic reagent may also be used in the purification schemes of thepresent invention, such as Chaps, zwitterion 3-14, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulf-onate. It iscontemplated also that urea may be added with or without anotherdetergent or surfactant.

Lysis or homogenization solutions may further contain other agents, suchas reducing agents. Examples of such reducing agents includedithiothreitol (DTT), beta.-mercaptoethanol, DTE, GSH, cysteine,cysteamine, tricarboxyethyl phosphine (TCEP), or salts of sulfurousacid. Once obtained, the nucleic acid is denatured by any method knownin the art to produce single stranded nucleic acid templates and a pairof first and second oligonucleotides is hybridized to the singlestranded nucleic acid template such that the first and secondoligonucleotides flank a target region on the template.

In some embodiments, nucleic acids may be fragmented or broken intosmaller nucleic acid fragments. Nucleic acids, including genomic nucleicacids, can be fragmented using any of a variety of methods, such asmechanical fragmenting, chemical fragmenting, and enzymatic fragmenting.Methods of nucleic acid fragmentation are known in the art and include,but are not limited to, DNase digestion, sonication, mechanicalshearing, and the like (J. Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 1989, 2.sup.nd Ed., Cold Spring Harbour LaboratoryPress: New York, N.Y.; P. Tijssen, “Hybridization with Nucleic AcidProbes—Laboratory Techniques in Biochemistry and Molecular Biology(Parts I and II)”, 1993, Elsevier; C. P. Ordahl et al., Nucleic AcidsRes., 1976, 3: 2985-2999; P. J. Oefner et al., Nucleic Acids Res., 1996,24: 3879-3889; Y. R. Thorstenson et al., Genome Res., 1998, 8: 848-855).U.S. Patent Publication 2005/0112590 provides a general overview ofvarious methods of fragmenting known in the art.

Genomic nucleic acids can be fragmented into uniform fragments orrandomly fragmented. In certain aspects, nucleic acids are fragmented toform fragments having a fragment length of about 5 kilobases or 100kilobases. In a preferred embodiment, the genomic nucleic acid fragmentscan range from 1 kilobases to 20 kilobases. Preferred fragments can varyin size and have an average fragment length of about 10 kilobases.However, desired fragment length and ranges of fragment lengths can beadjusted depending on the type of nucleic acid targets one seeks tocapture. The particular method of fragmenting is selected to achieve thedesired fragment length. A few non-limiting examples are provided below.

Chemical fragmentation of genomic nucleic acids can be achieved using anumber of different methods. For example, hydrolysis reactions includingbase and acid hydrolysis are common techniques used to fragment nucleicacid. Hydrolysis is facilitated by temperature increases, depending uponthe desired extent of hydrolysis. Fragmentation can be accomplished byaltering temperature and pH as described below. The benefit of pH-basedhydrolysis for shearing is that it can result in single-strandedproducts. Additionally, temperature can be used with certain buffersystems (e.g. Tris) to temporarily shift the pH up or down from neutralto accomplish the hydrolysis, then back to neutral for long-term storageetc. Both pH and temperature can be modulated to affect differingamounts of shearing (and therefore varying length distributions).

Other methods of hydrolytic fragmenting of nucleic acids includealkaline hydrolysis, formalin fixation, hydrolysis by metal complexes(e.g., porphyrins), and/or hydrolysis by hydroxyl radicals. RNA shearsunder alkaline conditions, see, e.g. Nordhoff et al.,Nucl. Acid. Res.,21 (15):3347-57 (2003), whereas DNA can be sheared in the presence ofstrong acids.

An exemplary acid/base hydrolysis protocol for producing genomic nucleicacid fragments is described in Sargent et al. (1988) Methods Enzymol.,152:432. Briefly, 1 g of purified DNA is dissolved in 50 mL 0.1 N NaOH1.5 mL concentrated HCl is added and the solution is mixed quickly. DNAwill precipitate immediately, and should not be stirred for more than afew seconds to prevent formation of a large aggregate. The sample isincubated at room temperature for 20 minutes to partially depurinate theDNA. Subsequently, 2 mL 10 N NaOH (OH—concentration to 0.1 N) is added,and the sample is stirred until the DNA re-dissolves completely. Thesample is then incubated at 65 degrees C. for 30 minutes in order tohydrolyze the DNA. Resulting fragments typically range from about250-1000 nucleotides but can vary lower or higher depending on theconditions of hydrolysis.

In one embodiment, after genomic nucleic acid has been purified, it isre-suspended in a Tris-based buffer at a pH between 7.5 and 8.0, such asQiagen's DNA hydrating solution. The re-suspended genomic nucleic acidis then heated to 65 C and incubated overnight. Heating shifts the pH ofthe buffer into the low- to mid- 6 range, which leads to acidhydrolysis. Over time, the acid hydrolysis causes the genomic nucleicacid to fragment into single-stranded and/or double-stranded products.

Chemical cleavage can also be specific. For example, selected nucleicacid molecules can be cleaved via alkylation, particularlyphosphorothioate-modified nucleic acid molecules (see, e.g., K. A.Browne, “Metal ion-catalyzed nucleic Acid alkylation and fragmentation,”J. Am. Chem. Soc. 124(27):7950-7962 (2002)). Alkylation at thephosphorothioate modification renders the nucleic acid moleculesusceptible to cleavage at the modification site. See I. G. Gut and S.Beck, “A procedure for selective DNA alkylation and detection by massspectrometry,” Nucl. Acids Res. 23(8):1367-1373 (1995).

Methods of the invention also contemplate chemically shearing nucleicacids using the technique disclosed in Maxam-Gilbert Sequencing Method(Chemical or Cleavage Method), Proc. Natl. Acad. Sci. USA. 74:560-564.In that protocol, the genomic nucleic acid can be chemically cleaved byexposure to chemicals designed to fragment the nucleic acid at specificbases, such as preferential cleaving at guanine, at adenine, at cytosineand thymine, and at cytosine alone.

Mechanical shearing of nucleic acids into fragments can occur using anymethod known in the art. For example, fragmenting nucleic acids can beaccomplished by hydroshearing, trituration through a needle, andsonication. See, for example, Quail, et al. (November 2010) DNA:Mechanical Breakage. In: eLS. John Wiley & Sons, Chichester.doi:10.1002/9780470015902.a0005 333.pub2.

The nucleic acid can also be sheared via nebulization, see (Roe, B A,Crabtree. J S and Khan, A S 1996); Sambrook & Russell, Cold Spring HarbProtoc 2006. Nebulizing involves collecting fragmented DNA from a mistcreated by forcing a nucleic acid solution through a small hole in anebulizer. The size of the fragments obtained by nebulization isdetermined chiefly by the speed at which the DNA solution passes throughthe hole, altering the pressure of the gas blowing through thenebulizer, the viscosity of the solution, and the temperature. Theresulting DNA fragments are distributed over a narrow range of sizes(700-1330 bp). Shearing of nucleic acids can be accomplished by passingobtained nucleic acids through the narrow capillary or orifice (Oefneret al., Nucleic Acids Res. 1996; Thorstenson et al., Genome Res. 1995).This technique is based on point-sink hydrodynamics that result when anucleic acid sample is forced through a small hole by a syringe pump.

In HydroShearing (Genomic Solutions, Ann Arbor, Mich., USA), DNA insolution is passed through a tube with an abrupt contraction. As itapproaches the contraction, the fluid accelerates to maintain thevolumetric flow rate through the smaller area of the contraction. Duringthis acceleration, drag forces stretch the DNA until it snaps. The DNAfragments until the pieces are too short for the shearing forces tobreak the chemical bonds. The flow rate of the fluid and the size of thecontraction determine the final DNA fragment sizes.

Sonication is also used to fragment nucleic acids by subjecting thenucleic acid to brief periods of sonication, i.e. ultrasound energy. Amethod of shearing nucleic acids into fragments by sonication isdescribed in U.S. Patent Publication 2009/0233814. In the method, apurified nucleic acid is obtained placed in a suspension havingparticles disposed within. The suspension of the sample and theparticles are then sonicated into nucleic acid fragments.

An acoustic-based system that can be used to fragment DNA is describedin U.S. Pat. Nos. 6,719,449, and 6,948,843 manufactured by Covaris Inc.U.S. Pat. No. 6,235,501 describes a mechanical focusing acousticsonication method of producing high molecular weight DNA fragments byapplication of rapidly oscillating reciprocal mechanical energy in thepresence of a liquid medium in a closed container, which may be used tomechanically fragment the DNA.

Another method of shearing nucleic acids into fragments uses ultrasoundenergy to produce gaseous cavitation in liquids, such as shearing withDiagonnode's BioRuptor (electrical shearing device, commerciallyavailable by Diagenode, Inc.). Cavitation is the formation of smallbubbles of dissolved gases or vapors due to the alteration of pressurein liquids. These bubbles are capable of resonance vibration and producevigorous eddying or microstreaming. The resulting mechanical stress canlead to shearing the nucleic acid in to fragments.

Enzymatic fragmenting, also known as enzymatic cleavage, cuts nucleicacids into fragments using enzymes, such as endonucleases, exonucleases,ribozymes, and DNAzymes. Such enzymes are widely known and are availablecommercially, see Sambrook, J. Molecular Cloning: A Laboratory Manual,3rd (2001) and Roberts R J (January 1980). “Restriction and modificationenzymes and their recognition sequences,” Nucleic Acids Res. 8 (1):r63-r80. Varying enzymatic fragmenting techniques are well-known in theart, and such techniques are frequently used to fragment a nucleic acidfor sequencing, for example, Alazard et al, 2002; Bentzley et al, 1998;Bentzley et al, 1996; Faulstich et al, 1997; Glover et al, 1995;Kirpekar et al, 1994; Owens et al, 1998; Pieles et al, 1993; Schuette etal, 1995; Smirnov et al, 1996; Wu & Aboleneen, 2001; Wu et al, 1998a.

The most common enzymes used to fragment nucleic acids areendonucleases. The endonucleases can be specific for either adouble-stranded or a single stranded nucleic acid molecule. The cleavageof the nucleic acid molecule can occur randomly within the nucleic acidmolecule or can cleave at specific sequences of the nucleic acidmolecule. Specific fragmentation of the nucleic acid molecule can beaccomplished using one or more enzymes in sequential reactions orcontemporaneously.

Restriction endonucleases recognize specific sequences withindouble-stranded nucleic acids and generally cleave both strands eitherwithin or close to the recognition site in order to fragment the nucleicacid. Naturally occurring restriction endonucleases are categorized intofour groups (Types I, II III, and IV) based on their composition andenzyme cofactor requirements, the nature of their target sequence, andthe position of their DNA cleavage site relative to the target sequence.Bickle T A, Krüger D H (June 1993), “Biology of DNA restriction,”Microbiol. Rev. 57 (2): 434-50; Boyer H W (1971). “DNA restriction andmodification mechanisms in bacteria”. Annu. Rev. Microbiol. 25: 153-76;Yuan R (1981). “Structure and mechanism of multifunctional restrictionendonucleases”. Annu. Rev. Biochem. 50: 285-319. All types of enzymesrecognize specific short DNA sequences and carry out the endonucleolyticcleavage of DNA to give specific fragments with terminal 5′-phosphates.The enzymes differ in their recognition sequence, subunit composition,cleavage position, and cofactor requirements. Williams R J (2003).“Restriction endonucleases: classification, properties, andapplications”. Mol. Biotechnol. 23 (3): 225-43.

Where restriction endonucleases recognize specific sequencings indouble-stranded nucleic acids and generally cleave both strands, nickingendonucleases are capable of cleaving only one of the strands of thenucleic acid into a fragment. Nicking enzymes used to fragment nucleicacids can be naturally occurring or genetically engineered fromrestriction enzymes. See Chan et al., Nucl. Acids Res. (2011) 39 (1):1-18.

In some embodiments, DNA is sheared in biological processes within anorganism, or a biological medium. Such DNA, or cell-free DNA, circulatesfreely in the blood stream. For example, cell-free tumor DNA (ctDNA) istumor DNA that circulates freely in the blood stream. Some embodimentsuse fragmented or sheared DNA, however, the DNA is obtained infragmented form.

In preferred embodiments of the present invention, the strands of duplexnucleic acid fragments are joined together in a complex, for example,see FIG. 1. Any linking molecule may be used to join the molecules. Thelinker used in the present invention may be synthesized or obtainedcommercially from various companies, for example, Integrated DNATechnologies, Inc., Gene Link, Inc., and TriLink Biotechnologies, Inc.The linker may be any molecule to join two primers or two nucleic acidfragments. The linking molecule may also join multiple fragmentstogether. Any number of fragments may be incorporated to the complex.

FIG. 1 illustrates a droplet based method of the invention for creatinglinked duplex nucleic acids from the sense and antisense strands of anucleic acid fragment. As shown, a double stranded cell-free DNA (cfDNA)having a rare variant on represented on both strands can be obtained.The double stranded template may then be added to an emulsion with oneor more gene specific forward primers (e.g., the emulsion may containmultiplexed forward and reverse primers specific to more than one geneor part of a gene), one or more gene specific reverse primers, auniversal linked primer. The emulsion may be subjected to emulsion PCRto create linked, duplex products. The emulsion can then be broken andunlinked template digested. The remaining, linked duplex products maythen be sequenced. Because double stranded product enters droplet, withforward and reverse gene-specific primers, duplex sequence informationmay be obtained. The linked products of the emulsion PCR contain bothtemplate senses at least about 50% of time, which lowers average errorrate. As shown in FIG. 1, a PCR error is introduced into the duplexproduct during the emulsion PCR but, because the PCR error is onlypresent on one strand and the true variant is present on both, the twocan be easily differentiated from each other during sequencing.

FIGS. 2 and 3 show exemplary universal linked primers and forward andreverse gene specific primers and methods for their use in PCRamplification to create linked duplex products. Preferably, the ampliconlength is kept short to improve sensitivity. In the examples, the targetregion between primers is about 86 bp. Additional gene specific primersare shown in FIG. 4.

FIGS. 5 and 6 illustrate sequencing methods of the invention using theproducts derived from FIGS. 2 and 3 respectively. The linked primers maycontain two or more sites and may be made of PEG, Traptavidin bound tobiotinylated DNA, DNA coated beads, DNA-coated nanoparticles, DNA-linkedto gel based beads (e.g., acrylamide). Beads may be polystyrene, latex,magnetic, silica, ferromagnetic or similar materials. Attachment can beby conventional methods and preferably by a combination of amino andcarboxyl groups.

Methods of the invention may include duplex identification strategiesfor droplet formed linked duplex molecules. As noted, droplet basedmethods of the invention may result in at least a 50% rate of linkedduplex fragment formation (linked molecules that contain representationsfrom each side of the DNA duplex) so, identification of those productsbecomes important in order to omit data from non-duplex products andreap the accuracy increasing benefits of the duplex products. Duplexidentification methods may include, for example, a two-stage PCRapproach using two sets of primers with different annealing temperatureswhere several initial cycles are performed at low temperature withgene-specific barcoding primers to amplify and identify each sense ofthe duplex, while adding a universal tail for subsequent cycles. Thenumber of barcoding cycles is limited to prevent labeling each sense ofthe duplex with multiple barcodes.

Subsequent cycles may then be performed at high temperature viauniversal primers because the barcoding primers are unable to bind underthose conditions. Duplex products may then be identified by the presenceof their sense specific barcodes during sequencing analysis.

FIGS. 14A-D illustrate duplex identification methods according tocertain embodiments of the invention. In the illustrated example, thefollowing may be added to the droplet: a linking primer; a universalforward primer and a universal reverse primer, each having a high Tm (Tmmay be increased using LNA); a barcoded forward gene specific primer anda barcoded reverse gene specific primer, each having a lower Tm and at alower concentration than the universal forward primer; and the duplextemplate. Emulsion PCR may then be run with a first cycle having a lowannealing temperature to allow the barcoded primers to bind the templatefollowed by a second low annealing temperature cycle to produce theproducts shown in FIG. 14B. A third low annealing temperature cycleallows the first cycle of universal primer binding. In this cycle,barcoded primers will still bind [A+B] to form more or the C and Dproducts, and may also bind C and D products to form more E and Fproducts.

After the third cycle, the products shown in FIG. 14C may be present inthe emulsion, which may then be subjected to a 4th low annealingtemperature cycle to allow a second cycle of universal primer binding.At the end of cycle 4, molecules with the full forward and reverseuniversal tails may be obtained as shown in FIG. 14C. The annealingtemperature may be increased for subsequent cycles. There may be some Iand J type products having different barcodes (e.g., they have the fulluniversal tails on either the forward or reverse side). They can onlyamplify linearly at a higher annealing temp.

The subsequent PCR cycles (5+) may have an increased annealingtemperature only allowing binding of universal primers to amplicons witha full universal tail as shown in FIG. 14D. The last few cycles may beat a low annealing temperature to allow the linking of amplified strandsvia a portion of the forward universal tail. Alternatively, a longerlinked primer may be used with the full forward universal tail whichallows linking at higher annealing temperatures but is harder tosynthesize and may be less efficient in linking. Linking top or bottomsense occurs at random so 50% of linked molecules using this linkedprimer should have 1 of each (duplex info). Linked primers with morethan 2 sites, for example 100 sites on a nanoparticle, on averagecontain duplex information nearly 100% of the time.

In certain embodiments, linked duplex molecules may be created withoutthe use of emulsion PCR. In non-droplet embodiments, a singleamplification cycle may be used to create a linked duplex moleculehaving both the sense and antisense strands of the original fragment.The linked duplex molecule may then be directly loaded in a flow cellfor sequencing, thereby avoiding amplification induced sequence orlength biases or (e.g., in whole genome sequencing) as well as avoidingamplification introduced errors and nucleic acid losses from poorloading efficiency. For example, where loading efficiency of a sequencercan be defined as: (number of output reads)/ (number of input moleculesable to form reads), the loading efficiency for the Illumina MiSeq is<0.1%, and is similar for other Illumina instruments. This is largelydue to fluidic losses, since over 600 uL of sample is loaded into thesequencer, while only ˜7 uL is retained inside the flow cell forbinding, resulting in large losses of starting material. Thenon-droplet, direct load methods described herein remedy theseinefficiencies. Methods of the invention may include a simplifiedworkflow that creates duplex molecule with one cycle of PCR. The duplexmolecules can then be used to seed a single cluster and provide highaccuracy sequencing reads. By loading the flow cell directly and thensequencing, DNA losses through loading are minimized.

Direct load, non-droplet methods of the invention have applicationsincluding whole genome sequencing where a small mass of DNA is present,but high accuracy is desired, such as tissue biopsy, needle aspirates,or small volume blood draws. Additional applications may include thosewhere DNA is degraded or damaged, such as in formalin-fixed,paraffin-embedded (FFPE) samples.

FIGS. 7A, 7B, 8A, and 8B show non-droplet linked duplex formationmethods according to certain embodiments of the invention. One (FIG. 7)or two (FIG. 8) linking adapters are ligated onto the double strandedgenomic template and then extended using a strand displacing polymeraseto create the linked duplex molecule. The linked duplex may then bedirectly loaded to a flow cell for sequencing. In two linking adapterapplications such as illustrated in FIG. 8, linked fragments may beformed in two orientations (i.e., linked fragments having the linker onone end and linked fragments having the linker on the opposite end). Asshown in FIGS. 7B and 8B, ligation may result in about 50% the desired,linked duplex product where other undesired products will not formclusters.

In various embodiments, the linked adapter ligation techniques describedherein may be applied to double stranded adapters as shown in FIGS. 7Aand 8A to help ensure that two different adapters ligate to a single DNAmolecule where ligation of the first adapter to one end of the genomictemplate brings the second adapter into close proximity, increasing theprobability of the second adapter ligating onto the other end of thetemplate.

FIG. 34 illustrates linked double stranded adapters ligated to atemplate where one of the double stranded adapters is a linking-typeadapter as used in FIG. 7A to create a linked template molecule throughstrand displacement. FIGS. 35 and 36 show linked double strandedadapters including two linking adapters as shown in FIGS. 8A and 8B forcreating two linked template molecules through strand displacement.FIGS. 35 and 36 show alternative connection points for linkersconnecting the two double stranded adapters.

Linked adapter ligation methods may be used to increase ligationefficiency over traditional ligation methods. FIG. 37 depicts linkedY-adapters ligated to template DNA. Even when ligating two of the sameY-adapters, because one side binding brings the second adapter intoclose proximity with the other side of the template, both ends of thetemplate are likely to bind the Y-adapters. Linked adapter ligation mayalso be used on single stranded DNA.

FIGS. 9A and 9B illustrate steps of a direct loading sequencing methodusing linked duplex molecules. In the exemplary method of FIGS. 9A and9B, a flow cell is initialized with reagents. A small volume linkedlibrary is then denatured and the whole volume loaded onto theinitialized flow cell. The flow cell ports are then sealed and thetemplate such as created in the methods illustrated in FIGS. 7 and 8, isbound to the flow cell. The DNA on the flow cell is extended and thenthe flow cell is loaded on the flow cell sequencing instrument.Exemplary flow cell binding is illustrated in FIG. 10 including thesteps of flow cell capture, extension, washing off of linked template,bridge amplification, and sequencing. Binding for the other sense strandof linked duplex template is analogous to that illustrated in FIG. 10.

FIG. 11 illustrates an exemplary off-line seeding protocol compared to adefault protocol. In certain embodiments, steps of the off-line seedingprotocol may include performing the following steps at the bench at roomtemperature: flush with LDR×5, flush with PR2×5, flush with HT1, loadTMP, and seal ports with PCR tape, where flush means filling the flowcell with the specified reagent, waiting about 10 seconds, and thenemptying the flow cell. After sealing the ports with PCR tape, the flowcell is incubated in a bead bath at 75 degrees Celsius for 10 minutes,followed by incubation at 40 degrees Celsius for 10 minutes. Returningthe flow cell to the bench at room temperature, the seal is removed, andthe flow cell is flushed with PR2 at 40 degrees Celsius 5 times, flushedwith AMS1 2 times, flushed with AMS1 with a two minute incubation at 40degrees Celsius 3 times, filled with AMS1, and transferred to a MiSeqinstrument (commercially available from Illumnia, Inc, San Diego,Calif.) for sequencing. Additional steps in the preparation protocol mayinclude taking the flow cell out from its plastic housing, pre-cuttingPCR tape for sealing ports, and protecting the flow cell from scratchesfrom bead bath, with PCR tape or scotch tape on both sides.

Linked ligation adapters of the invention may be used for target captureand selective amplification of target templates. Linked ligationadapters may be used with single stranded DNA (ssDNA) or, in certainembodiments, may be used with double stranded DNA (dsDNA). FIG. 30 showsan exemplary use of linked ligation adapters of the invention. Linkedligation adapters include adapters that may be sequencing adapters orcomprise universal priming sites and are linked to target sequencespecific probes. The probes are complimentary to at least a portion ofthe target template ssDNA. The probes bind the template ssDNA strand,bringing their linked adapter into close proximity to the template andallowing for ligation of the adapters to the ends of the ssDNA template.The universal priming sites in the ligated adapters then allow for PCRamplification of the target template using universal PCR withoutamplifying off target nucleic acids. This results in a targeted libraryincluding sequencing adapters and ready for sequencing.

FIGS. 38A-D show an exemplary method of double stranded linked ligation.Double stranded linked ligation takes advantage of isothermalrecombinase and single stranded binding proteins to generate strandinvasion of dsDNA allowing primers or probes to pair with complementarysequences in the dsDNA with the single stranded binding proteins thenbinding to displaced DNA strands to prevent the primer or probe frombeing displaced. The process is similar to that used in RecombinasePolymerase Amplification (RPA) as described in Piepenburg, O., et al.,2006, DNA Detection Using Recombination Proteins, PLoS Biol 4(7): e204,incorporated herein by reference. Methods allow for ligation of specificDNA targets based on recognition sequence. Target capture can beintegrated directly into ligation steps and makes for a simple targetednext generation sequencing workflow. The dsDNA methods described hereindirects ligation to a desired end of the DNA, allowing differentadapters to be added to each end. The methods allow for ligation of twodifferent adapters to a single template with high efficiency. Forexample, a y adapter and a hairpin could be ligated (one option shown inFIG. 38C), so that duplex information could be integrated with everysequencing read (as shown in FIG. 38D).

As shown in FIG. 38A, ligation adapters may be incubated with ligationprobes and recombinase to create a ligation complex. Ligation complexescan be made as separate parts and then linked together. For example, theligation probe can be linked to the ligation adapter before incubatingwith recombinase. Linkers may comprise, for example, PEG, regular DNAbases, modified DNA bases, or inverted DNA bases. The linker may benon-extendable to prevent extension of the ligation probe. The ligationprobe may be blocked to prevent extension Linking can occur throughclick chemistry, biotin/streptavidin binding, or other DNA linkingchemistries.

When the ligation complex is incubated with single stranded bindingprotein and a dsDNA genomic template comprising a target sequencecomplementary to the ligation probe, the ligation complex can bind thetarget sequence without requiring denaturing of the dsDNA as shown inFIG. 38B. Unbound ligation complexes may be optionally removed beforethe linked ligation adapters of the bound ligation complex are ligatedto the dsDNA template. The dsDNA template with ligated adapters can thenbe cleaned up and additional ligations may be performed as shown in FIG.38C. Additional ligations may be sequence specific, or standard, assequence specific binding sites should be ligated with high efficiency,and ligation will only occur at un-ligated ends. Second ligationadapters can be the same or different from the first. Additionally,ligation of adapters to each end of a target can also happen in a singlestep. Ligation can also be multiplexed to cover desired target regions.If increased target specificity is desired, target capture, such aslinked target capture, can be performed after ligation.

After adapter ligation to the target dsDNA, primers corresponding tosequences in the adapters (e.g., universal primers) may be used toamplify the target sequence for NGS using PCR amplification. Where ahairpin adapter has been ligated on one end of the dsDNA, duplexinformation can be obtained with each sequencing read as shown in FIG.38D. In high mass samples, PCR may not be required allowing for PCR-freetargeted ligation.

Linked adapters allow for ligation based on sequence recognition fordouble stranded or single stranded DNA targets. They further allow fortargeting of a single sense of DNA at a time. The linked adaptersdescribed herein allow for ligation to be directed to a desired end ofthe DNA. Linked adapters may find application in simple targetedsequencing and barcoding workflows, fusion detection, targeted PCR-freelibrary preparation, and droplet ligation and amplification.

FIG. 31 shows application of linked adapters to selectively capture andamplify fusion nucleic acids for fusion detection. Fusion genes resultfrom genomic rearrangements, such as deletions, amplifications andtranslocations. Such rearrangements can also frequently be observed incancer and have been postulated as driving event in cancer development.Accordingly, characterizing these fusion genes can provide importantinformation for personalized cancer diagnosis and treatment.

As shown in FIG. 31, an adapter is linked to a sequence specific probecomplementary to a portion of the fusion nucleic acid (ssDNA or RNA)that is known. The probe binds the target sequence, allowing the adapterto ligate to the end of the target sequence. The linker may becleavable, for example using a uracil digestion, and may be cleaved atthis stage. A second adapter linked to a probe complementary to the sameor a different portion of the known part of the fusion nucleic acid canthen be introduced allowing the probe to bind the target nucleic acidand bring the linked adapter into close proximity to ligate onto theother end of the fusion even though the sequence is unknown. The adapterligated template may then be amplified using universal primers and PCRto create a library for sequencing. This is useful in identifying andcharacterizing fusions where potentially only one side of the breakpoint is known. The described method is faster and cheaper thantraditional target capture and works better with RNA.

Linked ligation techniques may be used for surface capture as well toprepare flow cells for sequencing analysis. These techniques allow forcapturing of target molecules based on sequence followed by ligation tothe surface of the flow cell or other solid support. FIGS. 32A and 32Billustrate such a method. A flow cell is provided having an adapterbound to its surface in close proximity to surface bound and/or linkedtarget specific probe complementary to a portion of the target templatesequence. Additional free floating linked adapter/probe molecules areadded to the flow cell along with a sample including strands of thetarget DNA. The targeted DNA binds to the capture probe on the flow celland the free floating linked adapter/probe molecule. Unbound DNA andcontaminants can then be washed away from the flow cell leaving just thetarget or targeted DNA. ssDNA ligase can then be added and, due to theclose proximity of the surface bound adapter and the free floating probelinked adapter, the two adapters will ligate to the ends of the targetDNA leaving a flow cell surface bound target DNA with sequencingadapters that is ready for flow cell sequencing after denaturing theprobes and washing. Workflows are simplified by combining the ligation,target capture, and flow cell binding steps into one. A whole fragmentcan be sequenced as capture probes do not block sequencing. Thesemethods can be used with single molecule sequencers such as thoseavailable from Direct Genomics (Shenzhen, China) or NanoStringtechnologies (Seattle, Wash.).

In certain embodiments, two adapters may be linked together to increaseligation efficiency and to help ensure that two different adapters areligated to a single DNA molecule (as opposed to a DNA molecule with twoof the same adapters ligated). FIGS. 33A-33C illustrate a method ofligating linked adapters to a DNA molecule. A double stranded genomictemplate is provided and exposed to two double stranded adapters thatare linked together by, for example, PEG, nucleic acids, or other means.The linker may optionally be cleavable. The adapters may be joined bybound complementary sequences having a melting temperature (T_(m)) thatis high enough that the adapters remain linked during ligation but canbe denatured after ligation to separate the link. Once one adapter hasligated to the genomic template, the likelihood of the second, linkedadapter binding the other end of the template is very high due to itsclose proximity as shown in FIG. 33B. As shown in FIG. 33C, the secondadapter is ligated, ensuring that two different adapters ligate to thesame double stranded DNA molecule. The ligation linker can be optionallycleaved at this point for subsequent PCR amplification using, forexample, universal primers corresponding to primer sites included in theligated adapters.

For direct loading embodiments as well as other applications where theyield of flow cell loading and target capture yield are important, itmay be beneficial to combine flow cell loading with targeted sequencing,to minimize loss. Such a combination additionally simplifies theworkflow by eliminating an extra step. While methods exist for targetcapture on the flow cell, they suffer from at least two downsides.First, they are not able to sequence the region that is captured on theflow cell. For short fragments such as cell free DNA, this can amount toa large loss of signal. Secondly, they are unable to capture linkedduplex molecules, as described in the invention, for sequencing.Accordingly, methods of the invention include flow cell based targetcapture of duplex molecules. According to methods of the invention, theflow cell contains one sense of oligonucleotides (oligos) having targetregions, while the other sense are hair-pinned and not immediatelyavailable for binding. See FIG. 12. After one sense of linked moleculesis captured on the flow cell, the other flow cell oligos are activatedto capture the other sense of the linked fragments (e.g., using a uracildigest, enzyme digestion, or light). The template may then be extendedand cluster generation may continue as normal. In certain embodimentsthe one set of oligos may be complementary to the sense or antisensestrand of the duplex nucleic acid while the another set is complementaryto a universal adapter that has been attached to both the sense andantisense strands and the universal adapter oligos may be hair-pinned toprevent binding in an initial exposure step.

FIGS. 13A-E illustrate steps of an exemplary method for flow cell basedtarget capture of duplex molecules. FIG. 13A shows an exemplary targetcapture step where a linked molecule is loaded onto a flow cell, eitherdirectly or by conventional methods. FIG. 13B shows an exemplary step ofbinding the template to the flow cell where the linked molecule binds toa complementary capture region, and the other sense of flow cell oligosare released to bind both free ends of linked fragment. FIG. 13C showsan exemplary strand displacement step where strand displacing polymeraseis used to extend both fragment to create a doubly-seeded cluster. Thelinked template may then be denatured and removed from the flow cell asshown in FIG. 13D. Bridge amplification may then occur as normal, butwith two molecules seeding the cluster as shown in FIG. 13E.

Direct loading techniques of the invention may be used in whole genomesequencing applications without flow cell target capture steps with oneor two linking adapters. In targeted sequencing applications, afterligation with one or two linked adapters, a tube-based target capturetechnique may be used that is optimized for yield (e.g., having pooroff-target rejection but high yield). The linked duplex template maythen be directly loaded into the flow cell as described above with orwithout the target capture steps described in FIGS. 13A-E. In certainembodiments the intermediate tube-based target capture step may beomitted.

In certain embodiments, the linking molecule may be a streptavidinmolecule and the fragments to be linked may comprise biotinylatednucleic acid. In embodiments where linked primers are used to create thelinked nucleic acid fragments through amplification, the primers may bebiotinylated and joined together on a streptavidin molecule. Forexample, 4 fragments may be joined together on a tetramer streptavidin.More than four molecules could be joined through the formation ofconcatemers, for example. In certain methods of the invention, two ormore nucleic acid fragments may be linked through click chemistryreactions. See Kolb, et al., Click Chemistry: Diverse Chemical Functionfrom a Few Good Reactions, Angew Chem Int Ed Engl. 2001 Jun.1;40(11):2004-2021, incorporated herein by reference.

Linking molecules, for example and of several known nanoparticles, maylink large numbers of fragments including hundreds or thousands offragments in a single linked molecule. One example of a linkingnanoparticle may be polyvalent DNA gold nanoparticles comprisingcolloidal gold modified with thiol capped synthetic DNA sequences ontheir surface. See, Mirkin, et al., 1996, A DNA-based method forrationally assembling nanoparticles into macroscopic materials, Nature,382:607-609, incorporated herein by reference. The surface DNA sequencesmay be complimentary to the desired template molecule sequences or maycomprise universal primers.

The linking molecule may also serve to separate the nucleic acidfragments. In preferred embodiments, the fragments are oriented toprevent binding there between. With the linker creating spatialseparation and orientation of the fragments controlled, collapsing orbinding between the fragments can be avoided and prevented.

In some embodiments the linkers may be polyethylene glycol (PEG) or amodified PEG. A modified PEG, such as DBCO-PEG₄, or PEG-11 may be usedto join the two adapters or nucleic acids. In another example,N-hydroxysuccinimide (NHS) modified PEG is used to join the twoadapters. See Schlingman, et al., Colloids and Surfaces B: Biointerfaces83 (2011) 91-95. Any oligonucleotide or other molecule may be used tojoin adapters or nucleic acids.

In some embodiments, aptamers are used to bind two adapters or nucleicacids. Aptamers can be designed to bind to various molecular targets,such as primers or nucleic acids. Aptamers may be designed or selectedby the SELEX (systematic evolution of ligands by exponential enrichment)method. Aptamers are nucleic acid macromolecules that specifically bindto target molecules. Like all nucleic acids, a particular nucleic acidligand, i.e., an aptamer, may be described by a linear sequence ofnucleotides (A, U, T, C and G), typically 15-40 nucleotides long. Insome preferred embodiments, the aptamers may include inverted bases ormodified bases. In some embodiments, aptamers or modified apatmers,include at least one inverted base or modified base.

It should be appreciated that the linker may be composed of invertedbases, or comprise at least one inverted base. Inverted bases ormodified bases may be acquired through any commercial entity. Invertedbases or modified bases are developed and commercially available.Inverted bases or modified bases may be incorporated into othermolecules. For example, 2-Aminopurine can be substituted in anoligonucleotide. 2-Aminopurine is a fluorescent base that is useful as aprobe for monitoring the structure and dynamics of DNA.2,6-Diaminopurine (2-Amino-dA) is a modified base can form threehydrogen bonds when base-paired with dT and can increase the Tm of shortoligos. 5-Bromo-deoxyuridine is a photoreactive halogenated base thatcan be incorporated into oligonucleotides to crosslink them to DNA, RNAor proteins with exposure to UV light. Other examples of inverted basesor modified bases include deoxyUridine (dU), inverted dT,dideoxycytidine (ddC), 5-methyl deoxyCytidine, or 2′-deoxylnosine (dI).It should be appreciated that any inverted or modified based can be usedin linking template nucleic acids.

In preferred embodiments, the linker comprises a molecule for joiningtwo primers or two nucleic acid fragments. The linker may be a singlemolecule, or a plurality of molecules. The linker may comprise a fewinverted bases or modified bases, or entirely inverted bases or modifiedbases. The linker may comprise a both Watson-Crick bases and inverted ormodified bases.

It should be appreciated that any spacer molecule or linking moleculemay be used in the present invention. In some embodiments, the linker orspacer molecule may be a lipid or an oligosaccharide, or anoligosaccharide and a lipid. See U.S. Pat. No. 5,122,450. In thisexample, the molecule is preferably a lipid molecule and, morepreferably, a glyceride or phosphatide which possesses at least twohydrophobic polyalkylene chains.

The linker may be composed of any number of adapters, primers, andcopies of fragments. A linker may include two identical arms, where eacharm is composed of binding molecules, amplification primers, sequencingprimers, adapters, and fragments. A linker may link together any numberof arms, such as three or four arms. It should be appreciated that insome aspects of the invention, nucleic acid templates are linked by aspacer molecule. The linker in the present invention may be any moleculeor method to join two fragments or primers. In some embodiments,polyethylene glycol or a modified PEG such as DBCO-PEG₄ or PEG-11 isused. In some embodiments the linker is a lipid or a hydrocarbon. Insome embodiments a protein may join the adapters or the nucleic acids.In some embodiments, an oligosaccharide links the primers or nucleicacids. In some embodiments, aptamers link the primers or nucleic acids.When the fragments are linked, the copies are oriented to be in phase soto prevent binding there between.

In certain embodiments, a linker may be an antibody. The antibody may bea monomer, a dimer or a pentamer. It should be appreciated that anyantibody for joining two primers or nucleic acids may be used. Forexample, it is known in the art that nucleoside can be made immunogenicby coupling to proteins. See Void, B S (1979), Nucl Acids Res 7,193-204. In addition, antibodies may be prepared to bind to modifiednucleic acids. See Biochemical Education, Vol. 12, Issue 3.

The linker may stay attached to the complex during amplification. Insome embodiments, the linker is removed prior to amplification. In someembodiments, a linker is attached to a binding molecule, and the bindingmolecule is then attached to an amplification primer. When the linker isremoved, the binding molecule or binding primer is exposed. The exposedbinding molecule also attaches to a solid support and an arch is formed.The linker may be removed by any known method in the art, includingwashing with a solvent, applying heat, altering pH, washing with adetergent or surfactant, etc.

Methods of the invention provide for nucleic acids to be linked togetherwith a linker molecule. In samples with low genetic material, nucleicacids can be linked together in order to ensure that the duplexfragments amplified simultaneously or sequentially. Samples such asprenatal samples have low genetic content and amplifying duplexfragments increases the detectable content. This method reduces thesignal to noise ratio, improving the detection of the target sequence.

Methods of the invention utilize amplification to amplify a targetnucleic acid, such as a fragment, to a detectable level. It should beappreciated that any known amplification technique can be used in thepresent invention. Further, the amplified segments created by anamplification process may be themselves, efficient templates forsubsequent amplifications.

Amplification refers to production of additional copies of a nucleicacid sequence and is generally carried out using polymerase chainreaction or other technologies well known in the art (e.g., Dieffenbachand Dveksler, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y. [1995]). The amplification reaction may be anyamplification reaction known in the art that amplifies nucleic acidmolecules, such as polymerase chain reaction, nested polymerase chainreaction, ligase chain reaction (Barany F. (1991) PNAS 88:189-193;Barany F. (1991) PCR Methods and Applications 1:5-16), ligase detectionreaction (Barany F. (1991) PNAS 88:189-193), transcription basedamplification system, nucleic acid sequence-based amplification, rollingcircle amplification, and hyper-branched rolling circle amplification.

In some embodiments, multiple displacement amplification (MDA), anon-PCR based DNA amplification technique, rapidly amplifies minuteamounts of DNA samples for genomic analysis. The reaction starts byannealing random hexamer primers to the template: DNA synthesis iscarried out by a high fidelity enzyme at a constant temperature.However, it should be appreciated that any amplification method may beused with the current invention.

In certain embodiments of the invention, the amplification reaction isthe polymerase chain reaction. Polymerase chain reaction (PCR) refers tomethods by K. B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202, herebyincorporated by reference) for increasing concentration of a segment ofa target sequence in a mixture of genomic DNA without cloning orpurification. The process for amplifying the target sequence includesintroducing an excess of oligonucleotide primers to a DNA mixturecontaining a desired target sequence, followed by a precise sequence ofthermal cycling in the presence of a DNA polymerase. The primers arecomplementary to their respective strands of the double stranded targetsequence.

In some aspects of the invention, PCR primers are joined by a linkermolecule and through the PCR process, copies of both strands of a duplexfragment are linked to the primers. In other embodiments, adapters areadded to the primers or copies of the fragments. The resulting complexincludes, generally, one sense and one antisense strand of a duplexfragment directly or indirectly joined by a linking molecule. It shouldbe appreciated that one or both of the linked strand copies may includean error. However, there is a low probability that each fragment willhave a matching error at the exact same base. Disagreement between thetwo fragments at a base would indicate an error as opposed to a truevariant. The base could then be identified as an unknown, just from theraw sequencing data.

Primers can be prepared by a variety of methods including but notlimited to cloning of appropriate sequences and direct chemicalsynthesis using methods well known in the art (Narang et al., MethodsEnzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68:109 (1979)).Primers can also be obtained from commercial sources such as OperonTechnologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.The primers can have an identical melting temperature. The lengths ofthe primers can be extended or shortened at the 5′ end or the 3′ end toproduce primers with desired melting temperatures. Also, the annealingposition of each primer pair can be designed such that the sequence and,length of the primer pairs yield the desired melting temperature. Thesimplest equation for determining the melting temperature of primerssmaller than 25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)).Computer programs can also be used to design primers, including but notlimited to Array Designer Software (Arrayit Inc.), Oligonucleotide ProbeSequence Design Software for Genetic Analysis (Olympus Optical Co.),NetPrimer, and DNAsis from Hitachi Software Engineering. The TM (meltingor annealing temperature) of each primer is calculated using softwareprograms such as Oligo Design, available from Invitrogen Corp.

In some embodiments, to effect amplification, a mixture is denatured andthe primers then annealed to their complementary sequences within thetarget molecule. Following annealing, the primers are extended with apolymerase so as to form a new pair of complementary strands. The stepsof denaturation, primer annealing and polymerase extension can berepeated many times (i.e., denaturation, annealing and extensionconstitute one cycle; there can be numerous cycles) to obtain a highconcentration of an amplified segment of a desired target sequence. Thelength of the amplified segment of the desired target sequence isdetermined by relative positions of the primers with respect to eachother, and therefore, this length is a controllable parameter.

In some embodiments, to create complexes of the invention, primers arelinked by a linking molecule or a spacer molecule to create two linkedcopies of the fragment. In other embodiments, two fragments are linkedtogether following at least one PCR step. It should be appreciated thatPCR can be applied to fragments before or after the fragments are joinedvia a linking molecule. In some embodiments, when the fragments arejoined, PCR can be implemented on the joined fragments. In someembodiments, the linked copies undergo amplification. The amplificationstep includes linked primers. The result is that after a cycle of PCR,linked complexes comprising copies of the fragments are produced.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level that can be detected by severaldifferent methodologies (e.g., staining, hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications Amplified targetsequences can be used to obtain segments of DNA (e.g., genes) forinsertion into recombinant vectors.

Other amplification methods and strategies can also be utilized in thepresent invention. For example, another approach would be to combine PCRand the ligase chain reaction (LCR). Since PCR amplifies faster than LCRand requires fewer copies of target DNA to initiate, PCR can be used asfirst step followed by LCR. The amplified product could then be used ina LCR or ligase detection reaction (LDR) in an allele-specific mannerthat would indicate if a mutation was present. Another approach is touse LCR or LDR for both amplification and allele-specificdiscrimination. The later reaction is advantageous in that it results inlinear amplification. Thus the amount of amplified product is areflection of the amount of target DNA in the original specimen andtherefore permits quantitation.

LCR utilizes pairs of adjacent oligonucleotides which are complementaryto the entire length of the target sequence (Barany F. (1991) PNAS88:189-193; Barany F. (1991) PCR Methods and Applications 1:5-16). Ifthe target sequence is perfectly complementary to the primers at thejunction of these sequences, a DNA ligase will link the adjacent 3′ and5′ terminal nucleotides forming a combined sequence. If a thermostableDNA ligase is used with thermal cycling, the combined sequence will besequentially amplified. A single base mismatch at the junction of theoligonucleotides will preclude ligation and amplification. Thus, theprocess is allele-specific. Another set of oligonucleotides with 3′nucleotides specific for the mutant would be used in another reaction toidentify the mutant allele. A series of standard conditions could beused to detect all possible mutations at any known site. LCR typicallyutilizes both strands of genomic DNA as targets for oligonucleotidehybridization with four primers, and the product is increasedexponentially by repeated thermal cycling.

Amplification or sequencing adapters or barcodes, or a combinationthereof, may be attached to the fragmented nucleic acid. Such moleculesmay be commercially obtained, such as from Integrated DNA Technologies(Coralville, Iowa). In certain embodiments, such sequences are attachedto the template nucleic acid molecule with an enzyme such as a ligase.Suitable ligases include T4 DNA ligase and T4 RNA ligase, availablecommercially from New England Biolabs (Ipswich, Mass.). The ligation maybe blunt ended or via use of complementary overhanging ends.

In certain embodiments, following fragmentation, the ends of thefragments may be repaired, trimmed (e.g. using an exonuclease), orfilled (e.g., using a polymerase and dNTPs) to form blunt ends. In someembodiments, end repair is performed to generate blunt end 5′phosphorylated nucleic acid ends using commercial kits, such as thoseavailable from Epicentre Biotechnologies (Madison, Wis.). Upongenerating blunt ends, the ends may be treated with a polymerase anddATP to form a template independent addition to the 3′-end and the5′-end of the fragments, thus producing a single A overhanging. Thissingle A can guide ligation of fragments with a single T overhangingfrom the 5′-end in a method referred to as T-A cloning. Alternatively,because the possible combination of overhangs left by the restrictionenzymes are known after a restriction digestion, the ends may be leftas-is, i.e., ragged ends. In certain embodiments double strandedoligonucleotides with complementary overhanging ends are used.

In certain embodiments, one or more bar code is attached to each, any,or all of the fragments. A bar code sequence generally includes certainfeatures that make the sequence useful in sequencing reactions. The barcode sequences are designed such that each sequence is correlated to aparticular portion of nucleic acid, allowing sequence reads to becorrelated back to the portion from which they came. Methods ofdesigning sets of bar code sequences is shown for example in U.S. Pat.No. 6,235,475, the contents of which are incorporated by referenceherein in their entirety. In certain embodiments, the bar code sequencesare attached to the template nucleic acid molecule, e.g., with anenzyme. The enzyme may be a ligase or a polymerase, as discussed above.Attaching bar code sequences to nucleic acid templates is shown in U.S.Pub. 2008/0081330 and U.S. Pub. 2011/0301042, the content of each ofwhich is incorporated by reference herein in its entirety. Methods fordesigning sets of bar code sequences and other methods for attaching barcode sequences are shown in U.S. Pat. Nos. 7,537,897; 6,138,077;6,352,828; 5,636,400; 6,172,214; and 5,863,722, the content of each ofwhich is incorporated by reference herein in its entirety. After anyprocessing steps (e.g., obtaining, isolating, fragmenting,amplification, or barcoding), nucleic acid can be sequenced.

Exemplary methods for designing sets of barcode sequences and othermethods for attaching barcode sequences are shown in U.S. Pat. Nos.6,138,077; 6,352,828; 5,636,400; 6,172,214; 6235,475; 7,393,665;7,544,473; 5,846,719; 5,695,934; 5,604,097; 6,150,516; RE39,793;7,537,897; 6172,218; and 5,863,722, the content of each of which isincorporated by reference herein in its entirety.

The barcode sequence generally includes certain features that make thesequence useful in sequencing reactions. For example the barcodesequences can be designed to have minimal or no homopolymer regions,i.e., 2 or more of the same base in a row such as AA or CCC, within thebarcode sequence. The barcode sequences can also be designed so thatthey do not overlap the target region to be sequence or contain asequence that is identical to the target.

The first and second barcode sequences are designed such that each pairof sequences is correlated to a particular sample, allowing samples tobe distinguished and validated. Methods of designing sets of barcodesequences is shown for example in Brenner et al. (U.S. Pat. No.6,235,475), the contents of which are incorporated by reference hereinin their entirety. In certain embodiments, the barcode sequences rangefrom about 2 nucleotides to about 50; and preferably from about 4 toabout 20 nucleotides. Since the barcode sequence is sequenced along withthe template nucleic acid or may be sequenced in a separate read, theoligonucleotide length should be of minimal length so as to permit thelongest read from the template nucleic acid attached. Generally, thebarcode sequences are spaced from the template nucleic acid molecule byat least one base.

Methods of the invention involve attaching the barcode sequences to thetemplate nucleic acids. Template nucleic acids are able to be fragmentedor sheared to desired length, e.g. generally from 100 to 500 bases orlonger, using a variety of mechanical, chemical and/or enzymaticmethods. DNA may be randomly sheared via sonication, exposed to a DNaseor one or more restriction enzymes, a transposase, or nicking enzyme.RNA may be fragmented by brief exposure to an RNase, heat plusmagnesium, or by shearing. The RNA may be converted to cDNA before orafter fragmentation.

Barcode sequence is integrated with template using methods known in theart. Barcode sequence is integrated with template using, for example, aligase, a polymerase, Topo cloning (e.g., Invitrogen's topoisomerasevector cloning system using a topoisomerase enzyme), or chemicalligation or conjugation. The ligase may be any enzyme capable ofligating an oligonucleotide (RNA or DNA) to the template nucleic acidmolecule. Suitable ligases include T4 DNA ligase and T4 RNA ligase (suchligases are available commercially, from New England Biolabs). Methodsfor using ligases are well known in the art. The polymerase may be anyenzyme capable of adding nucleotides to the 3′ and the 5′ terminus oftemplate nucleic acid molecules. Barcode sequence can be incorporatedvia a PCR reaction as part of the PCR primer. Regardless of theincorporation of molecular barcodes or the location of the barcodes inthe event that they are incorporated, sequencing adaptors can beattached to the nucleic acid product in a bi-directional way such thatin the same sequencing run there will be sequencing reads from both the5′ and 3′ end of the target sequence. In some cases it is advantage touse the location of the barcode on the 5′ or 3′ end of the targetsequence to indicate the direction of the read. It is well known to oneskilled in the art how to attach the sequencing adaptors usingtechniques such as PCR or ligation.

FIG. 15 shows examples of possible configurations of adapters andprimers. As shown at 602, a P7 primer is attached to a Read2 primersite, which is attached to a complimentary region. At 603, a linked PCRpriming region is attached to a unique molecular identifier. As shown at604, a P5 primer is attached to an index read primer site, and a seedingcontrol site.

In some embodiments, multiple copies of a fragment are joined together.It should be appreciated that any number of fragments can be joinedtogether, whether 2, 3, 4, etc. The joined copies may be referred to asa unit. Several units may then be joined together with a linkingmolecule. It should be appreciated that any number of units may bejoined by a linking molecule. This increases the information densitywithin a complex. When the complex is attached to a solid support, thecomplex is amplified. The amplification products may be attached to thesolid support. By joining multiple copies of the fragment to the complexand then amplifying the complexes, information density on a solidsupport increases.

In certain embodiments, the nucleic acids may be amplified by two ormore joined primers. Any known method of amplification may be used inconjunction with the linked primers. In certain embodiments, digital PCRor emulsion PCR may be used to create two or more linked nucleic acidfragments for seeding sequencing clusters or for use in other sequencingmethods. In a preferred embodiment, a template nucleic acid may becreated by ligating adapters to a nucleic acid fragment of interest tobe sequenced. Adapters may optionally include universal priming sites,one or more sequencing primer sites, and unique cluster identifiers toensure that all sequencing reads in a given cluster originated from thesame starting template. For example, adapters may be used with varyingstem regions such as yl: CCTACTCGCTAC (SEQ ID No. 1), y2: ATGCGAGCCTCT(SEQ ID No. 2), y3: GCACCTCATCCA (SEQ ID No. 3), and y4: TGCAGGATGGTG(SEQ ID No. 4). Adapter sequences may include a unique clusteridentifier (UCI) which may comprise a series of random bases (e.g., 2,3, 4, 5, or more) to distinguish between neighboring clusters on asequencing flow cell. Adapter sequences may include aphosphorothioate-linked T in order to reduce 3′exonuclease digestionthat might remove T overhang and reduce ligation efficiency. A 3′phosphate blocker is optional but not essential for digital PCR methodsof the invention.

Once adapters have been ligated to the nucleic acid fragment to besequenced, an emulsion or droplet can be created. The droplets may beaqueous droplets surrounded by an immiscible carrier fluid. Methods offorming such droplets and conducting PCR amplification within thedroplets are shown for example in Link et al. (U.S. patent applicationnumbers 2008/0014589, 2008/0003142, and 2010/0137163), Stone et al.(U.S. Pat. No. 7,708,949 and U.S. patent application number2010/0172803), and Anderson et al. (U.S. Pat. No. 7,041,481 and whichreissued as RE41,780).

In preferred embodiments, a single template nucleic acid molecule isadded to a droplet in order to ensure that eventual clusters are seededwith only one template molecule and to prevent the formation of hybridlinked nucleic acid complexes containing different nucleic acidfragments. Various multiplex primers that may be gene specific are alsoadded to the droplet along with linked primers. The linked primers maybe two or more primers linked together according to any of the methodsdescribed herein. Linked primers may include, for example, universalpriming sites corresponding to the universal priming sites in theligated adapters as well as sequencing primer sites (e.g., differentindex priming sites to identify when more than one molecule has seeded acluster). In certain embodiments, the linked primers may include genespecific primers targeting specific regions of interest to be sequencedsuch that the initial ligation step may be avoided and an unmodifiednucleic acid fragment may be added directly to the droplet forlinked-primer digital PCR amplification. According to certain methods ofthe invention, the ligated template may comprise a priming sitecorresponding to the priming site of the linked universal primers andgene specific multiplex primers are used to create linked copies of thetarget nucleic acid.

Complexes of the invention may be attached to various solid supportssuch as microbeads, beads, channel walls, microchips, etc.

Sequencing the joined fragments may be by any method known in the art.The present invention has applications in various sequencing platforms,including the genome sequencers from Roche/454 Life Sciences (Margulieset al. (2005) Nature, 437:376-380; U.S. Pat. Nos. 6,274,320; 6,258,568;6,210,891), the SOLiD system from Life Technologies Applied Biosystems(Grand Island, N.Y.), the HELISCOPE system from Helicos Biosciences(Cambridge, Mass.) (see, e.g., U.S. Pub. 2007/0070349), and the Ionsequencers from Life Technologies Ion Torrent, Ion Torrent Systems, Inc.(Guilford, Conn.).

In preferred embodiments, sequencing is by methods where each base isdetermined sequentially. DNA sequencing techniques include classicdideoxy sequencing reactions (Sanger method) using labeled terminatorsor primers and gel separation in slab or capillary, sequencing bysynthesis using reversibly terminated labeled nucleotides,pyrosequencing, 454 sequencing, allele specific hybridization to alibrary of labeled oligonucleotide probes, sequencing by synthesis usingallele specific hybridization to a library of labeled clones that isfollowed by ligation, real time monitoring of the incorporation oflabeled nucleotides during a polymerization step, polony sequencing, andSOLiD sequencing. Sequencing of separated molecules has more recentlybeen demonstrated by sequential or single extension reactions usingpolymerases or ligases as well as by single or sequential differentialhybridizations with libraries of probes.

It should be appreciated that the linker may also be attached toadapters, primers, or binding molecules. The linker can be attached tothese species in any orientation or arrangement. The linking moleculemay be directly attached to an adapter or primer and indirectly linkedto the nucleic acid fragments. In some aspects of the invention, thelinking molecule is removed before or after amplification. In someembodiments, the linking molecule remains on the complex. In someembodiments, the linking molecule is removed prior to sequencing, wherein other embodiments the linking molecule remains on the complex duringsequencing.

A sequencing technique that can be used in the methods of the providedinvention includes, for example, Helicos True Single Molecule Sequencing(tSMS) (Harris T. D. et al. (2008) Science 320:106-109). In the tSMStechnique, a DNA sample is cleaved into strands of approximately 100 to200 nucleotides, and a polyA sequence is added to the 3′ end of each DNAstrand. Each strand is labeled by the addition of a fluorescentlylabeled adenosine nucleotide. The DNA strands are then hybridized to aflow cell, which contains millions of oligo-T capture sites that areimmobilized to the flow cell surface. The templates can be at a densityof about 100 million templates/cm². The flow cell is then loaded into aninstrument, e.g., HeliScope sequencer, and a laser illuminates thesurface of the flow cell, revealing the position of each template. A CCDcamera can map the position of the templates on the flow cell surface.The template fluorescent label is then cleaved and washed away. Thesequencing reaction begins by introducing a DNA polymerase and afluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are detected by imaging the flowcell surface. After imaging, a cleavage step removes the fluorescentlabel, and the process is repeated with other fluorescently labelednucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step. With thepresent invention, the linked fragments can be identified in tandem.Further description of tSMS is shown for example in Lapidus et al. (U.S.Pat. No. 7,169,560), Lapidus et al. (U.S. patent application number2009/0191565), Quake et al. (U.S. Pat. No. 6,818,395), Harris (U.S. Pat.No. 7,282,337), Quake et al. (U.S. patent application number2002/0164629), and Braslaysky, et al., PNAS (USA), 100: 3960-3964(2003), the contents of each of these references is incorporated byreference herein in its entirety.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is 454 sequencing (Roche) (Margulies,M et al. 2005, Nature, 437, 376-380). 454 sequencing involves two steps.In the first step, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. Using the methods of the present invention, joinedfragments as described above are captured on the beads. The joinedfragments attached to the beads are PCR amplified within droplets of anoil-water emulsion. The result is multiple copies of clonally amplifiedDNA fragments on each bead. In the second step, the beads are capturedin wells (pico-liter sized). Pyrosequencing is performed on each DNAfragment in parallel. Addition of one or more nucleotides generates alight signal that is recorded by a CCD camera in a sequencinginstrument. The signal strength is proportional to the number ofnucleotides incorporated. Pyrosequencing makes use of pyrophosphate(PPi) which is released upon nucleotide addition. PPi is converted toATP by ATP sulfurylase in the presence of adenosine 5′ phosphosulfate.Luciferase uses ATP to convert luciferin to oxyluciferin, and thisreaction generates light that is detected and analyzed.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is Ion Torrent sequencing (U.S. patentapplication numbers 2009/0026082, 2009/0127589, 2010/0035252,2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559),2010/0300895, 2010/0301398, and 2010/0304982), the content of each ofwhich is incorporated by reference herein in its entirety. In IonTorrent sequencing, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to a surface and is attached at a resolutionsuch that the fragments are individually resolvable. Using the methodsof the present invention, the joined fragments are attached to thesurface. Addition of one or more nucleotides releases a proton (H+),which signal detected and recorded in a sequencing instrument. Thesignal strength is proportional to the number of nucleotidesincorporated.

The invention also encompasses methods of sequencing amplified nucleicacids generated by solid-phase amplification. Thus, the inventionprovides a method of nucleic acid sequencing comprising amplifying apool of nucleic acid templates using solid-phase amplification andcarrying out a nucleic acid sequencing reaction to determine thesequence of the whole or a part of at least one amplified nucleic acidstrand produced in the solid-phase amplification reaction. Theinitiation point for the sequencing reaction may be provided byannealing of a sequencing primer to a product of a solid-phaseamplification reaction. In this connection, one or both of the adaptorsadded during formation of the template library may include a nucleotidesequence which permits annealing of a sequencing primer to amplifiedproducts derived by whole genome or solid-phase amplification of thetemplate library.

The products of solid-phase amplification reactions wherein both forwardand reverse amplification primers are covalently immobilized on thesolid surface are so-called bridged structures formed by annealing ofpairs of immobilized polynucleotide strands and immobilizedcomplementary strands, both strands being attached to the solid supportat the 5′ end. Arrays comprised of such bridged structures provideinefficient templates for typical nucleic acid sequencing techniques,since hybridization of a conventional sequencing primer to one of theimmobilized strands is not favored compared to annealing of this strandto its immobilized complementary strand under standard conditions forhybridization.

In order to provide more suitable templates for nucleic acid sequencing,it may be advantageous to remove or displace substantially all or atleast a portion of one of the immobilized strands in the bridgedstructure in order to generate a template which is at least partiallysingle-stranded. The portion of the template which is single-strandedwill thus be available for hybridization to a sequencing primer. Theprocess of removing all or a portion of one immobilized strand in a‘bridged’ double-stranded nucleic acid structure may be referred toherein as linearization, and is described in further detail in U.S. Pub.2009/0118128, the contents of which are incorporated herein by referencein their entirety.

Bridged template structures may be linearized by cleavage of one or bothstrands with a restriction endonuclease or by cleavage of one strandwith a nicking endonuclease. Other methods of cleavage can be used as analternative to restriction enzymes or nicking enzymes, including interalia chemical cleavage (e.g. cleavage of a diol linkage with periodate),cleavage of abasic sites by cleavage with endonuclease (for example‘USER’, as supplied by NEB, part number M55055), or by exposure to heator alkali, cleavage of ribonucleotides incorporated into amplificationproducts otherwise comprised of deoxyribonucleotides, photochemicalcleavage or cleavage of a peptide linker

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove the portion(s) of the cleaved strand(s)that are not attached to the solid support. Suitable denaturingconditions, for example sodium hydroxide solution, formamide solution orheat, will be apparent to the skilled reader with reference to standardmolecular biology protocols (Sambrook et al., supra; Ausubel et al.supra). Denaturation results in the production of a sequencing templatewhich is partially or substantially single-stranded. A sequencingreaction may then be initiated by hybridization of a sequencing primerto the single-stranded portion of the template. Thus, the inventionencompasses methods wherein the nucleic acid sequencing reactioncomprises hybridizing a sequencing primer to a single-stranded region ofa linearized amplification product, sequentially incorporating one ormore nucleotides into a polynucleotide strand complementary to theregion of amplified template strand to be sequenced, identifying thebase present in one or more of the incorporated nucleotide(s) andthereby determining the sequence of a region of the template strand.

Another example of a sequencing technology that can be used in themethods of the provided invention is Illumina sequencing. Illuminasequencing workflow is based on three steps: libraries are prepared fromvirtually any nucleic acid sample, amplified to produce clonal clustersand sequenced using massively parallel synthesis. Illumina sequencing isbased on the amplification of DNA on a solid surface using fold-back PCRand anchored primers. Genomic DNA is fragmented, and adapters are addedto the 5′ and 3′ ends of the fragments. DNA fragments that are attachedto the surface of flow cell channels are extended and bridge amplified.Using the methods of the present invention, the joined fragments areattached to the flow cell channels and extended and bridge amplified. Insome embodiments, the linker is removed prior to bridge amplification.In some embodiments, the linker remains attached to the fragments duringamplification. The fragments become double stranded, and the doublestranded molecules are denatured. Multiple cycles of the solid-phaseamplification followed by denaturation can create several millionclusters of approximately 1,000 copies of single-stranded DNA moleculesof the same template in each channel of the flow cell. Primers, DNApolymerase and four fluorophore-labeled, reversibly terminatingnucleotides are used to perform sequential sequencing. After nucleotideincorporation, a laser is used to excite the fluorophores, and an imageis captured and the identity of the first base is recorded. The 3′terminators and fluorophores from each incorporated base are removed andthe incorporation, detection and identification steps are repeated.Sequencing according to this technology is described in U.S. Pat. Nos.7,960,120; 7,835,871; 7,232,656; 7,598,035; 6,911,345; 6,833,246;6,828,100; 6,306,597; 6,210,891; U.S. Pub. 2011/0009278; U.S. Pub.2007/0114362; U.S. Pub. 2006/0292611; and U.S. Pub. 2006/0024681, eachof which are incorporated by reference in their entirety.

Methods of the present invention can be incorporated into the Illuminasequencing platform (commercially available from Illumina, Inc, SanDiego, Calif.). Using the present invention, libraries of linkedcomplexes comprising copies of both strands of a duplex fragment areprepared and then attached to the solid support. The complexes areamplified to produce clonal clusters and then sequenced using massivelyparallel synthesis. In this method, each cluster is seeded with onefragment. With the present invention, both strands of a duplex fragmentseed a cluster. During sequencing, if there is a lack of agreement at aparticular base between the amplicons, the error is detected.

The Illumina Genome Analyzer (detector, commercially available byIllumina) is based on parallel, fluorescence-based readout of millionsof immobilized sequences that are iteratively sequenced using reversibleterminator chemistry. In one example, up to eight DNA libraries arehybridized to an eight-lane flow cell. In each of the lanes,single-stranded library molecules hybridize to complementaryoligonucleotides that are covalently bound to the flow cell surface. Thereverse strand of each library molecule is synthesized and the nowcovalently bound molecule is then further amplified in a process calledbridge amplification. This generates clusters each containing more than1,000 copies of the starting molecule. One strand is then selectivelyremoved, free ends are subsequently blocked and a sequencing primer isannealed onto the adapter sequences of the cluster molecules.

Although the fluorescent imaging system is not sensitive enough todetect the signal from a single template molecule, the detector issensitive to detect the signal from each cluster. In this example of theinvention, the signals from numerous clusters are analyzed. Each clusteris expected to fluoresce at a value, for example, approximate to one ofthe four bases. If the cluster does not fluoresce at a value approximateto one of the four bases, then it is determined that an error exists atthat locus.

After sequencing, images are analyzed and intensities extracted for eachcluster. The Illumina base caller, Bustard, has to handle two effects ofthe four intensity values extracted for each cycle and cluster: first, astrong correlation of the A and C intensities as well as of the G and Tintensities due to similar emission spectra of the fluorophores andlimited separation by the filters used; and second, dependence of thesignal for a specific cycle on the signal of the cycles before andafter, known as phasing and pre-phasing, respectively. Phasing andpre-phasing are caused by incomplete removal of the 3′ terminators andfluorophores, sequences in the cluster missing an incorporation cycle,as well as by the incorporation of nucleotides without effective 3′terminators. Phasing and pre-phasing cause the extracted intensities fora specific cycle to consist of the signal of the current cycle as wellas noise from the preceding and following cycles.

Another example of a sequencing technology that can be used in themethods of the provided invention includes the single molecule,real-time (SMRT) technology of Pacific Biosciences. In SMRT, each of thefour DNA bases is attached to one of four different fluorescent dyes.These dyes are phospholinked. A single DNA polymerase is immobilizedwith a single molecule of template single stranded DNA at the bottom ofa zero-mode waveguide (ZMW). A ZMW is a confinement structure whichenables observation of incorporation of a single nucleotide by DNApolymerase against the background of fluorescent nucleotides thatrapidly diffuse in an out of the ZMW (in microseconds). It takes severalmilliseconds to incorporate a nucleotide into a growing strand. Duringthis time, the fluorescent label is excited and produces a fluorescentsignal, and the fluorescent tag is cleaved off. Detection of thecorresponding fluorescence of the dye indicates which base wasincorporated. The process is repeated. Using methods of the presentinvention, the process is repeated in tandem, with two fragments beinganalyzed.

Another example of a sequencing technique that can be used in themethods of the provided invention is nanopore sequencing (Soni G V andMeller A. (2007) Clin Chem 53: 1996-2001). A nanopore is a small hole,of the order of 1 nanometer in diameter Immersion of a nanopore in aconducting fluid and application of a potential across it results in aslight electrical current due to conduction of ions through thenanopore. The amount of current which flows is sensitive to the size ofthe nanopore. As a DNA molecule passes through a nanopore, eachnucleotide on the DNA molecule obstructs the nanopore to a differentdegree. Thus, the change in the current passing through the nanopore asthe DNA molecule passes through the nanopore represents a reading of theDNA sequence. Using methods of the present invention, two fragments areanalyzed simultaneously or sequentially, reducing the chance of anerror.

The present invention can be used with nanopore technology, such assingle molecule nanopore-based sequencing by synthesis (Nano-SBS). Thisstrategy can distinguish four bases by detecting 4 different sized tagsreleased from 5′-phosphate-modified nucleotides. As each nucleotide isincorporated into the growing DNA strand during the polymerase reaction,its tag is released and enters a nanopore in release order. Thisproduces a unique ionic current blockade signature due to the tag'sdistinct chemical structure, thereby determining DNA sequenceelectronically at single molecule level with single base resolution.Using the methods of the invention, both strands of a duplex fragmentcan be analyzed simultaneously or sequentially. See Kumar, et al.Scientific Reports, Article number 684, doi:10.1038/srep00684.

Functions described above such as sequence read analysis or assembly canbe implemented using systems of the invention that include software,hardware, firmware, hardwiring, or combinations of any of these.

One sequencing method which can be used in accordance with the inventionrelies on the use of modified nucleotides having removable 3′ blocks,for example as described in W004018497, US 2007/0166705A1 and U.S. Pat.No. 7,057,026, the contents of which are incorporated herein byreference in their entirety. Once the modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase cannot add further nucleotides. Once the nature of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides, it ispossible to deduce the DNA sequence of the DNA template. Such reactionscan be done in a single experiment if each of the modified nucleotideshas a different label attached thereto, known to correspond to theparticular base, to facilitate discrimination between the bases addedduring each incorporation step. Alternatively, a separate reaction maybe carried out containing each of the modified nucleotides separately.

Embodiments of the invention may incorporate modified nucleotides. Themodified nucleotides may be labeled (e.g., fluorescent label) fordetection. Each nucleotide type may thus carry a different fluorescentlabel, for example, as described in U.S. Pub. 2010/0009353, the contentsof which are incorporated herein by reference in their entirety. Thedetectable label need not, however, be a fluorescent label. Any labelcan be used which allows the detection of an incorporated nucleotide.One method for detecting fluorescently labeled nucleotides comprisesusing laser light of a wavelength specific for the labeled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means. Suitable instrumentation for recordingimages of clustered arrays is described in W007123744 and U.S. Pub.2010/0111768, the contents of which are incorporated herein by referencein their entirety.

FIGS. 18 and 19 illustrate alternative sequencing methods using systemsand methods of the invention. As shown in FIG. 18, after seedingclusters with multiple template copies and amplifying, errors can bedifferentiated from true variants through a drop in sequencing qualityin a single read at the position where the bases are not the same.Because all amplified strands in the cluster are all read at the sametime, in the same direction, a drop in signal quality is the only way todetermine a mixed base call within the cluster. In embodiments wherein acluster is seeded with multiple fragments such as both a sense andantisense strand or templates having different sequencing primer sites,true variants and errors may be identified by comparing results from twodifferent sequencing reads (e.g., reads from each sense or reads usingthe two different sequencing primers). FIG. 19 illustrates methods ofthe invention using two separate sequencing reads to compare base callsfrom a sense and antisense read. Sequencing or other introduced errorsshould only be seen on one of the reads while true variants should beobserved on both reads.

Linked target capture methods may include solution-based capture ofgenomic regions of interest for targeted DNA sequencing. FIGS. 20 and 21illustrate exemplary methods of solution-based target capture. Universalprobes and optional barcodes (which may be sense specific) are ligatedto extracted DNA. The ligated DNA product is then denatured and boundwith linked target capture probes comprising a universal priming siteand universal probe linked to a target specific probe. Target capture isperformed at a temperature where the universal probes cannot bind aloneunless local concentration is high due to the binding of the targetprobe. Strand displacing polymerase (e.g., BST, phi29, or SD) is thenused to extend the target-bound linked probes. The target probe isblocked from extension as indicated by the black diamond in FIGS. 20 and21 so that extension only occurs along the bound universal probe,copying the bound target nucleic acid strand that remains linked to thetarget probe. A number of linked-PCR extension cycles can then be usedto amplify the target sequences. PCR can then be performed usinguniversal primers corresponding to the universal priming sites from thelinked target capture probes to amplify one or both strands of thetarget nucleic acid. This PCR step can be performed in the same reactionwithout the need for a cleanup step. The amplified target sequence canthen be sequenced as described above. No gap is required between thelinked capture probes when used in opposite directions although a gap ispossible. The capture probes may be produced using universal 5′-linkersby joining the universal linkers to a pre-made capture probe. Thecapture probes can be joined by streptavidin/biotin or other means asdescribed above and the universal linker may be extended using thecapture probe as a template.

Methods of the invention include droplet based target capture,optionally using universal linked primers, to capture duplex molecules.The droplet based methods depicted in FIG. 22 are similar to thoseillustrated in FIG. 1 but use linked target capture probes as describedabove and depicted in FIGS. 20-21. Universal probes and optionalbarcodes (which may be sense specific) are ligated to extracted DNA(e.g., cell-free DNA). An emulsion is created as described above using aduplex template molecule and target capture probes comprising auniversal priming site and universal probe linked to a target specificprobe. As above, target capture is performed at a temperature where theuniversal probes cannot bind alone unless local concentration is highdue to the binding of the target probe and the capture probes areblocked from extending themselves but include a universal priming sitesuch that universal primers and linked universal primers included in theemulsion can be used to amplify the target nucleic acid to produce alinked duplex molecule comprising both sense and antisense strands ofthe target nucleic acid. Universal linkers may be omitted to performtarget capture alone. The emulsion can then be broken and un-linkedtemplate can be digested enzymatically leaving only linked duplexmolecules can then seed clusters or otherwise be sequenced as describedabove.

FIGS. 23A and B provide additional details of droplet-based targetcapture methods of the invention. Step 0 in FIG. 23A shows a duplextemplate molecule with universal probes and optional barcodes ligated toit is loaded into a droplet with linked and universal primers and targetcapture probes. The template DNA is denatured in the droplet and thetarget capture probes then bind the denatured template strands at atemperature where the universal probe will not bind alone unless thetarget probe is also bound. The universal primer then only binds tocaptured targets. Extension with strand displacing polymerase thenoccurs only on the captured targets. Moving to FIG. 23B, extensioncycles are then run (e.g., 4-6 cycles) until the liked target captureprobes and primers are exhausted. The resulting extension products arethen amplified using the universal linked primers to produce linkedduplex molecules with strand specific barcodes. As with thesolution-based methods, no gap is required between the linked captureprobes when in opposite directions. The linked capture probes can beused in one or both directions if omitting the universal linkers toperform target capture alone. Conventional polymerases can be mixed withstrand displacing polymerases within the droplet to carry out thevarious extension and amplification steps of the method.

Certain methods of the invention relate to target capture of linkedmolecules. Linked copies of molecules such as those created using themethods described above may be targeted and captured and converted tolinked molecules for sequencing. FIGS. 24-28 illustrate exemplarymethods of nanoparticle target capture of linked molecules. FIG. 24shows a nanoparticle having universal primers and a strand comprising atarget region complementary to a capture region of the linked moleculeto be captured. FIG. 25 illustrates binding of the capture region to thetarget region. This step occurs at a temperature where thetarget/capture regions will bind but the universal primers will not bindunless the capture region is bound. Unbound templates may be washed awayat this step. The temperature of the reaction may then be lowered topromote universal primer binding. FIG. 26 shows binding of the universalprimers to universal primer sites on the linked molecule. FIG. 27 showsuniversal primer extension by strand displacing polymerase to producenanoparticle linked copies of the target molecule comprising bothstrands of the original linked molecule. FIG. 28 shows a doubly seedednanoparticle that may be used to seed a cluster on a flow cell sequenceras described elsewhere in the application.

EXAMPLE 1 Sequencing Error Reduction in KRAS Amplicon Using DoubleSeeded Clusters

Flow cell clusters were seeded with single template molecules. Thesingle template copies were from a library of linked templates whereonly one of the linked template molecules was bound to the flow cell asshown in FIG. 17. The first 3000 singly-seeded clusters that aligned tothe KRAS amplicon were then analyzed for sequencing errors with anapplied quality threshold of greater than 35. The singly-seeded clustersresulted in a mean error of 0.13% with mean depth of about 3000 as shownin FIG. 16. Because the singly-seeded flow cell used a linked templatelibrary, the results may represent a lower error rate than would beexperienced using a standard single-seeding method with unlinkedtemplate molecules.

Flow cell clusters were then doubly seeded using linked templatemolecules where both of the linked molecules were bound to the flow cellto seed the cluster. The first 3000 doubly-seeded clusters that alignedto the chr12 amplicon were then analyzed for sequencing errors with thesame applied quality threshold of greater than 35 and a fluorescentchastity filter of 0.8 or greater. The doubly-seeded clusters provided a7-fold reduction in sequencing errors with less than 3% loss of analyzedbases over the singly-seeded clusters. The mean error rate for thedoubly-seeded clusters was 0.02% with a mean depth of about 2920 asshown in FIG. 29.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

What is claimed:
 1. A method of selectively ligating adapters to atarget nucleic acid, the method comprising: providing a first linkedligation adapter comprising a probe complimentary to a first portion ofa target nucleic acid, the probe linked to a first adapter comprising afirst universal priming site; exposing a sample comprising the targetnucleic acid to the first linked ligation adapter; ligating the targetnucleic acid to the first linked adapter; and amplifying the ligatedtarget nucleic acid by PCR using a first universal primer complimentaryto the first universal priming site.
 2. The method of claim 1, furthercomprising sequencing the target nucleic acid wherein the adapterfurther comprises a sequencing adapter.
 3. The method of claim 1 furthercomprising: providing a second linked ligation adapter comprising aprobe complimentary to a second portion of the target nucleic acid, theprobe linked to a second adapter comprising a second universal primingsite; and exposing the sample to the second linked ligation adapter;ligating the target nucleic acid to the second linked adapter whereinthe ligated target nucleic acid is amplified using the first universalprimer and a second universal primer complimentary to the seconduniversal priming site.
 4. The method of claim 3, wherein the sample issimultaneously exposed to the first and second linked ligation adapters.5. The method of claim 3, wherein the first and the second portion ofthe target nucleic acid are the same.
 6. The method of claim 5, whereinthe sample is exposed to the second linked ligation adapter after beingexposed to the first linked ligation adapter.
 7. The method of claim 1,wherein the target nucleic acid is a fusion nucleic acid.
 8. The methodof claim 7, wherein only a portion of the fusion nucleic acid is known.9. The method of claim 1, wherein the probe complimentary to the firstportion of the target nucleic acid is bound to a solid support proximateto the first adapter wherein the first adapter is also bound to thesolid support.
 10. The method of claim 9, further comprising: providinga second linked ligation adapter comprising a probe complimentary to asecond portion of the target nucleic acid, the probe linked to a secondadapter comprising a second universal priming site; and exposing thesample to the second linked ligation adapter; ligating the targetnucleic acid to the second linked adapter; wherein the sample isamplified using the first universal primer and a second universal primercomplimentary to the second universal priming site.
 11. The method ofclaim 10, further comprising washing the solid support to remove unboundnucleic acids present in the sample before amplification.
 12. The methodof claim 9, wherein the solid support is a flow cell.
 13. The method ofclaim 1, wherein the probe complimentary to the first portion of thetarget nucleic acid is linked to the first adapter by a linker selectedfrom the group consisting of a polyethylene glycol derivative, anoligosaccharide, a lipid, a hydrocarbon, a polymer, an inverted base,and a protein.
 14. The method of claim 13, wherein the linker iscleavable.
 15. The method of claim 1, wherein the target nucleic acid isdouble stranded DNA (dsDNA), wherein the first linked ligation adapterfurther comprises recombinase complexed with the probe and the firstadapter is a double stranded adapter, and wherein the sample comprisingthe target nucleic acid is exposed to the first linked ligation adapterin the presence of single stranded binding protein.
 16. The method ofclaim 1, wherein the adapter comprises a sequence of random nucleotides.17. The method of claim 1, wherein the adapter does not comprise auniversal priming site.
 18. The method of claim 1, wherein the targetnucleic acid is DNA or RNA.
 19. The method of claim 1, wherein theexposing, ligating, and amplifying steps are performed in a droplet.